Transgenic plants overexpressing a plant vacuolar H + -ATPase

ABSTRACT

Transgenic plants are described which are engineered to overexpress vacuolar H + -PPase. Plants such as tobacco and petunia transformed with  A. Thaliana  AVP-1 are shown to have increased meristematic activity resulting in larger leaves, stem, flower, fruit, root structures, increased salt tolerance, enhanced drought and freeze tolerance. Methods of making such plants are also described.

GOVERNMENT SUPPORT

The invention described herein was supported, in whole or in part, bygrants GM52414, DK54214, DK43495, DK51509, DK34854 and GM35010 from theNational Institutes of Health and by grant MCB9317175 from the NationalScience Foundation. The Government has certain rights in the invention.

RELATED APPLICATION(S)

This application is filed as a 371 of PCT/US01/09548 and claims priorityto U.S. patent application Ser. No. 09/644,039 filed Aug. 22, 2000, theentire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to genetically-altered plants that arehardy with respect to environmental stresses, such as drought and/orfreezing, oversized with respect to vegetative and/or sexual structure(as compared to their normal phenotypic counterparts), and capable ofgrowing in media of high salinity. Such plants also display highmeristematic activity, and increased in cellular division activity.

2. Background of the Related Art

The prospects for feeding humanity as we enter the new millennium areformidable. Given the every increasing world population, it remains amajor goal of agricultural research to improve crop yield. It also is amajor goal of horticultural research to develop non-crop plants whichare hardier, such as ornamental plants, grasses, shrubs, and otherplants found useful or pleasing to man.

Until recently crop and horticultural improvements depended on selectivebreeding of plants having desirable characteristics. Such selectivebreeding techniques, however, were often less than desirable as manyplants have within them heterogenous genetic complements that do notresult in identical desirable traits of their parents.

Advances in molecular biology have allowed mankind to manipulate thegermplasm of animals and plants. Genetic engineering of plants entailsthe isolation and manipulation of genetic material (typically in theform of DNA or RNA) and the subsequent introduction of that geneticmaterial into a plant. Such technology. has led to the development ofplants with increased pest resistance, plants that are capable ofexpressing pharmaceuticals and other chemicals, and plants that expressbeneficial traits. Advantageously such plants not only contain a gene ofinterest, but remain fertile.

One area of interest of late in plant sciences has been the developmentof plants with improved stress resistance. In general, plants possessand maintain adaptive mechanisms to ensure survival during periods ofadverse environmental conditions. Two commons stresses that plantscommonly encounter are freezing and drought, both of which areassociated with cellular dehydration. It is known that certain plantscontain genes turned on by exposure to cold or prolonged periods ofdehydration that encode for products that are directly or indirectlyresponsible for providing greater resistance to drought and/or freezethan many of their counterparts. A number of genes responsive to heatand water stress have now been characterized (See, e.g., U.S. Pat. Nos.5,837,545, 5,071,962, 4,707,359). These genes are believed by many toproduce certain proteins, such as “Water Stress Proteins”, that arepostulated to aid the plants survival. For example, certain plantsexposed to stress conditions produce a hormone called abcisic acid (ABA)which helps plants close their stromata, thereby reducing the severityof the stress. Unfortunately, ABA is known to inhibit the formation ofnew leaves, to cause flowers and fruit to drop off, and to lead to areduction in yield.

Most tropical plants are not believed to have evolved the ability totolerate prolonged drought and/or freezing. Conversely, many temperateplants are known to have developed at least some ability to toleratesuch conditions. The productivity of plant varieties in dry conditions,and after freeze, differ dramatically. For example, tobacco (Nicotianaspp.) produces fresh leaves that are highly sensitive to drought, andcannot be produced commercially in areas with a limited water supply andhigh degree of evaporation. In contrast to water stress, very little isknown about proteins and genes which participate in freezing tolerance.However, it has been hypothesized that a major component of freezetolerance may involve tolerance to dehydration (See, e.g., Yelenosky, G.C., Guy, L. (1989) Plant Physiol. 89: 444-451).

Another particular area of interest of late has been the development ofplants with improved abilities to grow in salinized soil. Salinizationof soil occurs when water supplies contain dissolved salt. Uponevaporation of water from such supplies, salts gradually accumulate inthe soil. The progressive salinization of irrigated land compromises thefuture of agriculture in many of the most productive areas of our planet(Serrano, R., et al., Crit. Rev. Plant Sci., 13:121-138 (1994)). Forexample, arid regions offer optimal photoperiod and temperatureconditions for the growth of most crops, but suboptimal rainfall.Artificial irrigation has solved the problem only in the short term asit has been found that soils in such environments frequently are rapidlysalinized. To grow in salinized environments, plants must maintain amuch lower ratio of Na⁺/K⁺ in their cytoplasm than that present in thesoil, preventing the growth of a number of plants, including food crops.

Physiological studies suggest that salt exclusion in the root, and/orsalt sequestration in the leaf cell vacuoles, are critical determinantsfor salt tolerance (Kirsch, M., et al., Plant Mol. Biol., 32:543-547(1996)). Toxic concentrations of sodium chloride (NaCl) build up firstin the fully expanded leaves where NaCl is compartmentalized in thevacuoles. Only after their loading capacity is surpassed, do thecytosolic and apoplasmic concentrations reach toxic levels, ultimatelyleading to loss of turgor, ergo plant death. It has been suggested thathyperacidification of the vacuolar lumen via the V-ATPase provides extraprotons required for a Na⁺/H⁺ exchange-activity leading to thedetoxification of the cytosol (Tsiantis, M. S., et al., Plant J.,9:729-736 (1996)). Salt stress is known to increase both ATP- andpyrophosphate (PPi)-dependent H⁺ transport in tonoplast vesicles of, forexample, sunflower seedling roots. Salt treatments also induce anamiloride-sensitive Na⁺/H⁺ exchange activity (Ballesteros, E., et al.,Physiologia Plantarum, 99:328-334 (1997)). In the halophyteMesembryanthemum crystallinum, high NaCl stimulates the activities ofboth the vacuolar H⁺-ATPase (V-ATPase) and a vacuolar Na⁺/H⁺ antiporterin leaf cells.

Yet another area of agricultural interest is to improve the yield ofcrop plants and to improve the aesthetic qualities of certain decorativeplants. The yield of a plant crop, and the aesthetics of certaindecorative plants, may be improved by growing plants that are largerthan the wild-type plant in vegetative and/or reproductive structure, aswell as improving the growth rate of plants.

A number of compounds have been touted in the prior art as improving therate of plant growth and biomass production in useful components of theplant. For example., cyclodextrins applied to tissue culture media hasbeen asserted to improve the rate of cell tissue culture growth bymechanisms including increased cell division (See, e.g., U.S. Pat. No.6,087,176). Certain plant growth hormones, such as auxins (whichpromote, among other things, root growth), cytokinins, and gibberellicacid (which promotes, among other things, stem growth) when applied toplant tissues are also known to promote increased cellular division. Italso is known in the art that certain growth factors may be used toincrease plant and/or plant flower size. Unfortunately, isolation andapplication of such growth hormones and factors is costly and timeconsuming.

U.S. Pat. No. 5,859,338 discloses that modification of the CLAVATA1 geneof Arabidopsis thaliana causes a loss normal control of cell division inshoot apical meristems and floral meristems. In either case, the loss ofcontrol is said to cause an enlargement of the meristem. In flowers, theenlargement is said to lead to an increase in the number of floralorgans, including an increase in carpel number, which increases fruitsize and seed number. U.S. Pat. No. 5,859,338 provides clavatal nucleicacids and proteins, and modified clavatal nucleic acids and proteins, toresult in altered meristem phenotypes.

U.S. Pat. No. 5,750,862 discloses a method for controlling plant cellgrowth comprising modulating the level and or catalytic activity of acell cycle control protein in the plant. In particular the patentdiscloses that by elevating levels of the protein p34^(cdc2),regeneration into plants of single or groups of cells can befacilitated. Control of regeneration may also be effectuated by controlof regulatory elements which indirectly result in modulation ofp34^(cdc2) activity.

A need, therefore, exists for plants having improved stress resistanceto drought and/or freeze, possessing larger size attributes thanwild-type counterpart varieties, and having increased tolerance to saltin the soil in which they are growing, and which provide for increasedbiomass of usefull components.

SUMMARY OF THE INVENTION

The present invention discloses a transgenic plant having upregulatedexpression of vacuolar pyrophosphatase. It has been found that plantsdisplaying such upregulated activity are generally larger than wild-typecounterparts, demonstrate improved stress resistance to drought and/orfreeze, have increased tolerance to salt in the media in which they aregrowing, and display higher meristematic activity (cell division)leading to greater biomass in certain plant parts as compared to wildtype plants.

Any suitable exogenous nucleic acid molecule which alters expression ofvacuolar pyrophosphatase in the plant can be used to transform thetransgenic plants in accord with the present invention. The exogenousnucleic acid can comprise nucleic acid that encodes a vacuolarpyrophosphatase protein (an exogenous vacuolar pyrophosphatase), such asAVP1, a functional portion thereof (peptide, polypeptide), or ahomologue thereof, and/or nucleic acid that alters expression of theendogenous vacuolar pyrophosphatase of the plant into which theexogenous nucleic acid is introduced. By “exogenous nucleic acid” it ismeant a nucleic acid from a source other than the plant cell into whichit is introduced, or into a plant or plant part from which thetransgenic part was produced. The exogenous nucleic acid used fortransformation can be RNA or DNA, (e.g., cDNA, genomic DNA). Inaddition, the exogenous nucleic acid can be circular or linear,double-stranded or single-stranded molecules. Single-stranded nucleicacid can be the sense strand or the anti-sense strand. By a “functionalportion” of a nucleic acid that encodes a vacuolar pyrophosphataseprotein it is meant a portion of the nucleic acid that encodes a proteinor polypeptide which retains a functional characteristic of a vacuolarpyrophosphatase protein. In a particular embodiment, the nucleic acidencodes AVP1, a functional portion or a homologue thereof. The AVP1nucleic acid may be obtained, for example, from Arabidopsis or any otherplant, or synthetically synthesized.

Nucleic acid that alters expression of the endogenous vacuolarpyrophosphatase of the plant into which the exogenous nucleic acid isintroduced includes regulatory sequences (e.g., inducible, constitutive)which function in plants and antisense nucleic acid. Examples ofregulatory sequences include promoters, enhancers. The nucleic acid canalso include, for example, polyadenylation site, reporter gene and/orintron sequences and the like whose presence may not be necessary forfunction or expression of the nucleic acid but can provide improvedexpression and/or function of the nucleic acid by affecting, forexample, transcription and/or stability (e.g., of mRNA). Such elementscan be included in the nucleic acid molecule to obtain optimalperformance of the nucleic acid.

The nucleic acid for use in the present invention can be obtained from avariety of sources using known methods. For example, the nucleic acidencoding a vacuolar pyrophosphatase (e.g., AVP1) for use in the presentinvention can be derived from a natural source, such as tobacco,bacteria, tomato or corn. In one embodiment, the nucleic acid encodes avacuolar pyrophosphatase that corresponds to a wild type of thetransgenic plant. In another embodiment, the nucleic acid encodes avacuolar pyrophosphatase that does not correspond to a wild type of thetransgenic plant. Nucleic acid that alters expression of the endogenousvacuolar pyrophosphatase of the plant into which the exogenous nucleicacid is introduced (e.g., regulatory sequence) can also be chemicallysynthesized, recombinantly produced and/or obtained from commercialsources.

A variety of methods for introducing the nucleic acid of the presentinvention into plants are known to those of skill in the art. Forexample, Agrobacterium-mediated plant transformation, particlebombardment, microparticle bombardment (e.g., U.S. Pat. Nos. 4,945,050;5,100,792) protoplast transformation, gene transfer into pollen,injection into reproductive organs and injection into immature embryoscan be used. The exogenous nucleic acid can be introduced into anysuitable cells(s) of the plant, such as root cell(s), stem cell(s)and/or leaf cell(s) of the plant.

Any suitable plant, including angiosperms, monocots and dicots, andgynmosperms, and algae can be used to produce the transgenic plants,tissue cultures or cell cultures of the present invention. For example,tomato, corn, tobacco, rice, sorghum, cucumber, lettuce, turf grass,ornamental (e.g., larger flowers, larger leaves) and legume plants canbe transformed as described herein to produce the transgenic plants ofthe present invention. In addition, the transgenic plants of the presentinvention can be grown in any medium which supports plant growth such assoil or water (hydroponically).

A transgenic plant of the present invention is preferably tolerant tohigh salt concentrations in soil. By the term “salt” it is meant toinclude any salt, that is a compound formed when hydrogen of an acid isreplaced by a metal or its equivalent, and includes, without limitation,salts comprising monovalent and divalent toxic cations, NaCl, KCl,CaCl₂, MgCl, CdCl, ZnCl, and sulfide salts.

Salt tolerance may be introduced into a plant of the present inventionby transforming plant cells with exogenous nucleic acid which alters theexpression of vacuolar pyrophosphatase in the plant such that expressionis upregulated. Any suitable vacuolar pyrophosphatase, several of whichhave been cloned, can be used in the compositions and methods of thepresent invention (e.g., Sarafian, V., et al., Proc. Natl. Acad. Sci.,USA, 89:1775-1779 (1992); Lerchl, J., et al., Plant Molec. Biol., 29:833-840 (1995); Kim, Y., et al., Plant Physiol., 106:375-382 (1994)). Ina particular embodiment, the present invention relates to a transgenicplant which is tolerant to salt comprising an exogenous nucleic acidconstruct which is designed to overexpress AVP1 (Sarafian, V., et al,Proc. Natl. Acad. Sci., USA, 89:1775-1779 (1992)). Transformation of theplant cells may be carried out in a whole plant, seeds, leaves, roots orany other plant part. Such transgenic plants are preferably altered suchthat they grow in a concentration of salt that inhibits growth of acorresponding non-transgenic plant. Transgenic progeny of the transgenicplants, seeds produced by the transgenic plant and progeny transgenicplants grown from the transgenic seed, which are also the subject of thepresent invention, advantageously carry such salt tolerant trait. Plantsmay be regenerated from transformed cells to yield transgenic plants,which may be screened for certain levels of salt tolerance. In apreferred embodiment, the exogenous nucleic acid encodes AVP1, or ahomologue thereof. Preferably expression of the vacuolar pyrophosphatasein the plant is enhanced to an extent that the transgenic plant istolerant to sodium chloride (NaCl) when the NaCl concentration is fromabout 0.2M to about 0.3M. A transgenic plant capable of growing in saltwater may also be produced by introducing into one or more cells of aplant nucleic acid which upregulates expression of vacuolarpyrophosphatase in the plant to yield transformed cells. As used herein,“salt water” includes water characterized by the presence of salt, andpreferably wherein the concentration of salt in the water is from about0.2M to about 0.4M. In one embodiment, salt water refers to sea water.

The transgenic plants of the present invention can also be used toproduce double transgenic plants which are tolerant to salt (about 0.2Mto about 0.4M salt concentration). In one embodiment, the presentinvention relates to a double transgenic plant which is tolerant to saltcomprising one or more plant cells transformed with exogenous nucleicacid which alters expression of a vacuolar pyrophosphatase and an Na⁺/H⁺antiporter in the plant. The vacuolar pyrophosphatase in an advantageousconstruct is AVP1, or a homologue thereof, and the Na⁺/H⁺ antiporter isAtNHX1, or a homologue thereof. The present invention also encompassestransgenic progeny of the double transgenic plant, as well as seedsproduced by the transgenic plant and a progeny transgenic plant grownfrom the seed.

Drought and/or freeze tolerance may also be introduced into plants bytransforming plant cells with exogenous nucleic acid which alters theexpression of vacuolar pyrophosphatase in the plant such that suchexpression is upregulated. In a preferred embodiment there is provided asubstantially drought and/or freeze resistant transgenic plant whichcomprises a genome having one or more exogenously introduced vacuolarH⁺-translocating pump genes. A particularly preferred fertile transgenicplant eliciting drought and/or freeze tolerance, as well as the abilityto grow in saline soils, comprises an isolated exogenous chimeric DNAconstruct encoding vacuolar H⁺-translocating pump, preferably operablylinked to a promoter, such as the 35-S promoter or any other strongpromoter, including, without limitation, tissue specific promoters. Thetransgenic plant may contain a polynucleotide sequence comprising anexogenous tonoplast pyrophosphate H⁺ pump gene operably linked to apromoter. In yet another particularly preferred drought and/or freezeresistant transgenic plant having the capacity to grow in saline soils,the polynucleotide sequence comprises an exogenous tonoplastpyrophosphate H⁺ pump gene operably linked to a double tandem enhancerof the 35S promoter. A particularly preferred tonoplast pyrophosphate H⁺pump gene is the AVP1 gene.

Upregulation of expression of vacuolar pyrophosphatase by the methodsdescribed above may also be used to provide a plant having largervegetative and/or sexual organs than wild type counterpart plants. Thatis, the present invention provides for a method of increasing the yieldof a plant comprising introducing into one or more cells of a plantnucleic acid which alters expression of vacuolar pyrophosphatase in theplant to yield transformed cells, thereby increasing the yield of theplant. The method can further comprise regenerating plants from thetransformed cells to yield transgenic plants and selecting a transgenicplant which is larger than its corresponding wild type plant, therebyproducing a transgenic plant which is larger than its corresponding wildtype plant. Also encompassed by the present invention is a method ofmaking a transgenic plant (e.g, an ornamental plant) having increasedflower size compared to its corresponding wild type plant comprisingintroducing into one or more cells of a plant nucleic acid which altersexpression of vacuolar pyrophosphatase in the plant to yield transformedcells.

Upregulation of expression of vacuolar pyrophosphatase by the methodsdescribed above may also be used to provide a plant with meristematicactivity cell division rate which is enhanced over wild-type counterpartplants. As would be recognized by one of ordinary skill in the art,meristems are central to higher plant development, as almost allpost-embryonic organs, including roots, leaves, flowers and axillarymeristems and cambium are initiated by either shoot or root meristems.Increased meristematic activity results in higher biomass in one or moreaspects of plant structure, as evidenced by dry weight of plantstructure, root structure as well as stem structure. Increasedmeristemic activity is hypothesized to result in increased rate of shootregeneration in root, leaf, hypocotyl, and cotyledon explants, andincreased overall plant growth rate.

The present inventor has discovered that overexpression of apyrophosphate driven proton (H⁺) pump at the vacuole leads to a greaterproton pumping capacity that results in a greater ion uptake into thevacuoles that lowers the osmotic potential of the cells, and also leadsto an increase in the capacity of plant cells to divide and multiply. Aswould be understood by one of ordinary skill in the art, such findingcan have great commercial importance, e.g., reducing time for wood, cornetc. production by transforming cells so as to overexpress such pumps,and enhancing shoot regeneration capacity in plants with poor or slowregeneration capacity, such as woody plants, crops, e.g. corn, andornametals e.g., orchids. Overexpression so as to produce such enhancedcell division and multiplication may be performed using any of theconstruct described herein. While a number of inducible promoters andtissue specific promoters may be used to trigger overexpression of thegene and/or homologues of the gene, a preferred construct includes atonoplast pyrophosphate driven H⁺ pump gene (AVP-1) operably linked to achimeric promoter (e.g., double tandem enhancer of 35S promoter)designed to overexpress AVP-1.

There is also disclosed in the present invention novel gene cassettesincluding cassettes comprising a tonoplast pyrophosphate driven H⁺ pumpgene operably linked to a chimeric promoter. A novel gene cassettecomprising an exogenous tonoplast pyrophosphate driven H⁺ pump geneoperably linked to a promoter, as well as novel coding sequencescomprising an exogenous tonoplast pyrophosphate driven H⁺ pump geneoperably linked to a double tandem enhancer of the 35S promoter.Preferably such coding sequence is designed to overexpress AVP1.

There is also disclosed in the present invention novel expressionvectors including an expression vector containing a polynucleotidesequence comprising a exogenous tonoplast pyrophosphate driven H⁺ pumpgene operably linked to a double tandem enhancer of the 35S promoter andfurther operatively linked to a multiple cloning site, and an expressionvector containing a polynucleotide sequence comprising a exogenoustonoplast pyrophosphate driven H⁺ pump gene operably linked to a doubletandem enhancer of the 35S promoter and further operatively linked to aheterologous coding sequence.

It is recognized by the present inventor, that the disclosed inventionmay have application to any plant, including, without limitation, cropplants, ornamental plants, grasses, shrubs, or any other plant founduseful or pleasing to man, including having application to monocots,dicots, angiosperms, gymnosperms, and algae.

BRIEF DESCRIPTION OF THE DRAWINGS

The above description, as well as further objects, features andadvantages present invention will be more fully understood withreference to the following detailed description when taken inconjunction with the accompanying drawings, wherein:

FIG. 1A is an overhead view of representative (out of 10 plants each)wild type (WT) and two independent transgenic lines (1′ and 2′) grownhydroponically for seven weeks on a 10 hour light/dark cycle;

FIGS. 1B(1), 1B(2) and 1B(3) are a photomicrographs of the root and roothairs of representative five day old seedlings obtained fromrepresentative WT, 1′ and 2′ of FIG. 1A grown parallel to the surface onvertical plant nutrient agar plates;

FIG. 1C is an immunoblot of membrane fractions isolated from wild type(WT) and two independent transgenic lines (1′ and 2′) overexpressingAVP-1;

FIG. 2 is an overhead view of a representative wild type plant (WT)versus representative transgenic plants overexpressing AVP-1 (1′ and 2′)after exposure to 7 days of water deficit stress.

FIG. 3 is a perspective view of wild-type plants (WT) versusrepresentative transgenic plants overexpressing AVP-1 (1′ and 2′) grownin salty soil.

FIG. 4A is a schematic representation of a working model of thetransporters involved in sodium sequestration at the yeast pre-vacuolarcompartment; Nhx1 (Na⁺/H⁺ antiporter), Vmal (vacuolar membraneH⁺-ATPAse), Gef1 (yeast CLC chloride channel), Ena1 (plasma membraneNa⁺-ATPase).

FIG. 4B is a schematic representation of a working model of thetransporters involved in sodium sequestration at the yeast pre-vacuolarcompartment shown in FIG. 4A, which also includes Avp1 (A. thalianavacuolar pyrophosphate-energized proton pump).

FIG. 5A and FIG. 5B are bar graphs showing the intracellular Na⁺ and K⁺contents of wild-type yeast strains and of yeast strains carryingvarious mutations affecting sodium tolerance wherein the values are themean of two determinations, and the bars represent the standarddeviations.

FIG. 14 is an overhead view of shoot regeneration from root andcotyledon explants from wild-type (T) and AVP-1 overexpressing (1′ and2′) Arabidopsis plants, cultured in the SIM medium of FIG. 13.

FIG. 7A is a bar graph of Na⁺ content of wild-type plants (WT) versusrepresentative transgenic plants overexpressing AVP-1 (1′ and 2′) grownin salty soil.

FIG. 7B is a bar graph of K+ content of wild-type plants (WT) versusrepresentative transgenic plants overexpressing AVP-1 (1′and 2′) grownin salty soil.

FIG. 8 is a graph of the uptake of calcium into the 35SAVP-1 transgenicvacuolar membrane vesicles (squares) of 2′ of FIG. 3 versus calciumuptake into vesicles obtained from wild type (WT) of FIG. 3.

FIGS. 9A and 9B are illustrations demonstrating the 35SAVP-1 theorizedmechanism for a higher accumulation of solids into vacuoles via a protondriven function versus that of WT vacuoles.

FIG. 10 is an overhead view of the leaf foliage of wild type versustransgenic (1′ and 2′) overexpressing Arabidopsis plants with leavespositioned with respect to one another according to size (X axis).

FIGS. 11A and 11B are overhead views of watered (distilled water) leavesfrom wild type and transgenic (1′ and 2′) Arabidopsis plantsoverexpressing AVP-1 demonstrating growth of root structures.

FIG. 12 is an overhead view of shoot regeneration in representative wildtype petunia leave cuttings (WT) versus representative transgenicpetunia plant leave cuttings overexpressing AVP-1 (35-S AVP-1).

FIG. 13 is an overhead view of shoot regeneration from 5-day oldcotyledons placed in SIM induction medium obtained from wild type (WT)and AVP-1 overexpressing (1′ and 2′) Arabidiopsis plants.

FIG. 14 is bar graph of osmotic potential in megapascals (MPa) of fullyhydrated leaves from WT and two AVP-1 overexpressing lines (1′ and 2′)from Arabidopsis plants. The leaf osmotic potential was measured byusing a Wescor (Logan, UT) 5500 osmometer.

FIG. 15 is an overhead view of petunia explants incubated on MS mediumshowing callus induction from the explants after six weeks ofincubation.

DETAILED DESCRIPTION OF THE INVENTION

Since plant vacuoles constitute 40 to 99% of the total intracellularvolume of a mature plant cell, changes in the size of the vacuole havedramatic effects upon cell size (R. G. Zhen, E. J. Kim, P. A. Rea, inThe Plant Vacuole. (Academic Press Limited, 1997), vol. 25, pp.298-337). The volume of the vacuole is controlled by ion and waterfluxes mediated by pumps and transporters. In plants the driving forcethat triggers the movement of ions, solutes and water across membranesis a proton gradient. The activity of the vacuolar H⁺-pumps results inluminal acidification and the establishment of a H⁺ electrochemicalpotential gradient across the vacuolar membrane, which powers thesecondary active transporters of inorganic ions, sugars, and organicacids. The activity of these transporters modulates cellular pH and ionhomeostasis and leads to the accumulation of solutes required togenerate the osmotic potential that promotes vacuolar expansion (H. Sze,X. Li, M. G. Palmgren, The Plant Cell 11, 677-689 (1999)).

There are three distinct pumps that generate proton electrochemicalgradients. One at the plasma membrane that extrudes H⁺ from the cell (PMH⁺-ATPase) and two at the vacuolar membrane or other endomembranecompartments that acidify their lumen (the vacuolar type H⁺-ATPase andH⁺-PPase) (R. A. Leigh, in The Plant Vacuole L. a. Sanders, Ed.(Academic Press, San Diego, Calif., 1997), vol. 25, pp. 171-194.).

Previous work has shown that a decrease in the levels of the A subunitof the vacuolar H⁺-ATPase of carrot, using an antisense construct,resulted in a plant with reduced cell expansion and altered leafmorphology (J. P. Gogarten, et al., The Plant Cell 4, 851-864 (1992)).The present inventor has hypothesized that an increased supply of H⁺into the vacuole could accelerate cell expansion. Recently, based on thetheory that as the availability of protons in the vacuolar function ofion accumulation, it has been hypothesized by the same inventor thataccumulation of solids in the vacuoles might be useful to protectagainst draught and to provide for a more freeze resistant plants.

The present inventor has recognized that plants have a number ofvacuolar H⁺-translocating pumps, and that by upregulating theiractivity, increasing their expression, upregulating their transcriptionand/or translation, or increasing their copy number that one canincrease accumulation of solids in the vacuole due to an increase in theavailability of protons in the vacuoles. The inventor tested thishypothesis by increasing the copy number of the vacuolarH⁺-translocating pump, the inorganic pyrophosphatase or V-PPase thatconsists of a single polypeptide (R. G. Zhen, E. J. Kim, P. A. Rea, inThe Platt Vacuole. (Academic Press Limited, 1997), vol. 25, pp.298-337). In Arabidopsis the V-PPase encoded by the AVP-1 gene iscapable of generating a H⁺ gradient across the vacuole membrane(tonoplast) similar in magnitude to that of the vacuolar H⁺-ATPase (V.Sarafian, Y. Kim, R. J. Poole, P. A. Rea, Proc. Natl. Acad. Sci. 89,1775-1779 (1992)). As would be understood by one of ordinary skill inthe art, similar genes in other plants should function in a similarmanner.

It is known that H⁺-PPase is the main proton pump of vacuolar membranesin growing tissue. The later may be due to the fact that in growingtissue, nucleic acids, DNA, RNAs, proteins and cellulose etc. areactively being synthesized for the construct of the new cells, and as aresult, a large amount of PPi is produced as a by-product of thesemetabolic processes. The energy stored in the PPi molecule may betransformed into a different source of energy, namely a H⁺-gradientacross the vacuolar membrane. This H⁺-gradient constitutes the drivingforce for the vacuolar accumulation of solutes that generate thesufficient osmotic differential that enables the plant cell to initiategrowth. While the present invention is not limited in any manner to anyparticular hypothesis for the increased growth effects seen, the presentinventor has hypothesized that in transgenic plants overexpressing AVP-1that the greater number of H⁺-PPiases has a positive effect on thevelocity of the generation of the H⁺ gradient, rendering a more activemeristem.

In one embodiment, a construct comprising a vacuolar pyrophosphatasegene operably linked to a promoter designed to overexpress the vacuolarpyrophosphatase (e.g., an expression cassette) is used to produce thetransgenic plants of the present invention. As used herein the term“overexpression” refers to greater expression/activity than occurs inthe absence of the construct. In a particular embodiment, a constructcomprising an AVP1 gene operably linked to a chimeric promoter designedto overexpress AVP1 is used to produce the transgenic plants of thepresent invention. More particularly, the present invention relates to aconstruct wherein the AVP1 gene is operably linked to a double tandemenhancer of a 35S promoter.

The transgenic plants of the present invention may find utility otherthan those associated with the food value or ornamental value,industrial value such as, for example, wood production. For example, thetransgenic plants of the present invention may uptake different or moreions than their wild-type counterparts. As discussed below, studies withmutant yeast strains (ena1) demonstrates that H⁺-translocating pumps atthe vacuole plays an important role in cation detoxification in higherplants (the plant components involved in an intracellular cationdetoxification system being identified by complementing salt-sensitivemutants of the budding yeast Saccharomyces cerevisiae). Transgenicplants and/or progeny thereof comprising exogenous nucleic acid whichalters expression of vacuolar pyrophosphatase in the plant in accordwith such studies may be used to bioremediate soil and growth medium.Such plants can be used to remove cations (e.g., monovalent and/ordivalent cations) from a medium which can support plant growth (e.g.,soil, water). For example, transformed plants of the present inventioncan be used to remove sodium (Na), lead (Pb), manganese (Mn) and/orcalcium (Ca) ions from a medium which supports plant growth.

To demonstrate the effect that an increased supply of H⁺ into thevacuole would have on resistance to drought and/or freeze, and toleranceto salt growth, as well as size of the plants, the present inventorgenerated transgenic plants containing extra copies of a vacuolar protonpump, AVP-1.

Arabidopsis thaliana plants were transformed with constructs containingthe AVP-1 gene. Transgenic lines containing extra copies of this genewere then isolated. The AVP-1, open reading frame was cloned into theXma1 site of a modified pRT103 [R. Topfer, V. Matzeit, B. Gronenborn, J.Schell and H -H. Steinbiss, Nucleic acid Research 15, 5890 (1987)]. Thisvector contains a tandem repeat of the 35-S promoter. A HindIII fragmentcontaining the 35-S tandem promoter, AVP-1 ORF and the polyadenylationsignal was subcloned into the HindIII site of the pPZP212 vector [P.Hajdukiewicz, Z. Svab and P. Maliga, Plant Molecular Biology 25, 989-994(1994)]. Agrobacterium-mediated transformation was performed via vacuuminfiltration of flowering Arabidopsis thaliana (ecotype columbia).Transgenic plants were selected by plating seeds of the transformedplants on plant nutrient agar plates supplemented with 25 mg/literkanamnycin. Plants were subsequently selected for two generations toidentify transgenic plant homozygous for the transgene.

FIG. 1A is an overhead picture of representative (out of 10 plants each)wild type (WT) and two independent transgenic lines (1′ and 2′) grownhydroponically for seven weeks on a 10 hour light/dark cycle. As can beseen in FIG. 1A, a visual comparison of transgenic line 2′, whichexpresses the AVP-1 protein at highest level, transgenic line 1′, andwild type (WT), demonstrates that the amount of AVP-1 correlates withthe size of the plants. The mass of the transgenic plants was found tobe greater than that of wild type. The dry weight of the entiretransgenic plants, measured after 24 hours at 75° C. (n=4), fortransgenic lines 1′ and 2′ was found to be 1.5 and 3 times greater thenthat of wild-type (WT).

FIG. 1B(1), FIG. 1B(2) and FIG. 1B(3) are photomicrographs(magnification: times 40; bar length on photograph=2 mm) of the root androot hairs of representative five day old seedlings obtained fromrepresentative WT, 1′transgenic and 2′ transgenic of FIG. 1A grownparallel to the surface on vertical plant nutrient agar plates.Seedlings of both transgenic lines 1′ and 2′ showed root hairs with anaverage length 40 and 70% larger than wild-type (WT) root hairs (FIG.1B) (Root hair length along the whole root was determined from fivemembers of each set of seedlings. An average of 80 root hairs per plantwere measured). The length of the root hairs is correlated with the sizeof the vacuole, so the increased size of the root hair is likely toresult from increased vacuolar volume. This compares with theArabidopsis mutant rdh3 which has been reported to have reduced vacuolarvolume and is a short plant with abnormally short root hairs (M. E.Galway, J. W. J. Heckman, J. W. Schiefelbein, Planta 201, 209-218(1997)). It is recognized that the increased root structure will have apositive impact on soil erosion, nitrogen fixation in legumes, and willaid in water and nutrient uptake by the plant.

FIG. 1C is an immunoblot of membrane fractions isolated from wild type(WT) and two independent transgenic lines (1′ and 2′) overexpressingAVP-1. Total membrane fractions were isolated from shoots of eight weekold wild type (WT) and AVP-1 transgenic plants (1′ and 2′) grown in ahydroponic media for 8 weeks. Homogenate of plant shoots weresequentially centrifuged for 15 and 30 min at 8,000 and 100,000 rpmsrespectively. The 100 mg membrane pellet was re-suspend in 10 mM Tris,pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol and 1 mM PMSF Protein (10ug) was separated on a 10% SDS-PAGE, electroblotted and immunostainedwith antibodies raised against a KLH-conjugated synthetic peptidecorresponding to the putative hydrophilic loop IV of the AVP-1 protein(V. Sarafian, Y. Kim, R. J. Poole, P. A. Rea, Proc. Natl. Acad. Sci. 89,1775-1779 (1992)). PPase was detected by chemiluminescence. FIG. 1Cillustrates that the transgenic lines (1′ and 2′) express AVP-1 proteinat higher levels than the wild type (WT) (that is, 1′=1.6 fold increaseand 2′=2.4 fold increase over WT).

As wheat that has been deprived of water is rendered more droughttolerant by an increase in cell K⁺ content (from 100 mM to 300 mM) (S.Gupta, G. Berkowitz, P. Pier, Plant Physiol 89, 1358-1365 (1989)), it ishypothesized (but the invention is not hereby limited by such theory)that the increased drought resistance of the AVP-1 transgenic plants maybe a consequence of their higher vacuolar concentration of potassiumthat results in a increased water retention capability. Laboratory testsappear to confirm this.

FIG. 2 is an overhead view of a representative wild type plant (WT)versus representative transgenic plants overexpressing AVP-1(1′ and 2′)after exposure to 7 days of water deficit stress. Wild type andtransgenic plants overexpressing AVP-1 (FIG. 3A) were tested for droughttolerance (24° C.). After 7 days of water deficit stress wild type (WT)plants withered, whereas plants from both 35S AVP-1 transgenic lines (1′and 2′) were turgid and alive. Furthermore, when the drought stressedplants were then watered, transgenic plants pursued normal growth,bolted and set seeds, whereas wild type plants died. The relative watercontent of leaves from wild type and 35SAVP-1 transgenic plants weredetermined along the water deficit stress, demonstrating increased waterretention by the transgenic lines as compared to the WT plants.

FIG. 14 is bar graph of osmotic potential of fully hydrated leaves fromwild-type (WT) and two AVP-1 overexpressing lines (1′ and 2′) fromArabidopsis plants. The decreased osmotic potential in leaves oftransgenic plants measured at a constant water content is consistentwith the contention that AVP-1 overexpression results in increasedsolute accumulation, and therefore enhancing water retention capability.

While not illustrated in the accompanying illustrations, similar resultsmay be seen with respect to freeze challenge (<0° C.) over a 24 hour ormore period for a number of plant species. While not limited to suchhypothesis, transgenic plants overexpressing AVP-1 (1′ and 2′) arebelieved to provide enhanced protection from freeze as compared to wildtype (WT) plants due to the higher amounts of cations in the vacuoles.Higher amounts of cations confer a greater osmotic pressure that leadsto a greater water retention capability endowing the plants not onlywith the ability to withstand low soil water potentials, but alsoproviding greater protection from freezing that leads to significantdesiccation of the plants.

FIG. 3 is a perspective view of wild type plants (WT) versusrepresentative transgenic plants overexpressing AVP-1 (1′ and 2′) grownin salty soil. Five wild-type plants (WT) and five of the two AVP-1overexpressing transgenic lines (1′ and 2′) were grown on soil in a 10hour light/dark cycle. Plants were watered with a diluted nutrientsolution (⅛ MS salts) for six weeks and subsequently watered with adiluted nutrient solution supplemented with NaCl. The concentration ofNaCl began with, 100 mM and was increased every four days by 50 mM. Theillustrations in FIG. 3 corresponds to representative plants at thetenth day in the presence of 300 mM NaCl. FIG. 3 illustrates that thetwo AVP-1 plant types (1′ and 2′) were significantly hardier in saltysoil as compared to wild-type plants. The fact that geneticallyengineered Arabidopsis thaliana plants that overexpress either AVP1 (thepyrophosphate-energized vacuolar membrane proton pump, this work) orAtNHX1 (the Na⁺/H⁺ antiporter, (Apse, M., et al., Science, 285:1256-1258(1999)) and this work) are capable of growing in the presence of highNaCl concentrations strongly supports the strategy described herein. Adouble transgenic plant would be expected to demonstrate a furtherenhanced salt-tolerant phenotype. These Arabidopsis thalianatransporters or their counterparts may perform similar function inimportant agricultural crops. The increased size of 35S AVP1 Arabidopsistransgenic plants also contribute to potential yield increases ingenetically engineered crops.

A Working Model of Cation Homeostasis in Plant Organelles

While the present invention is not limited to any particular hypothesis,the present inventor have developed a working model for cationhomeostasis in plant organelles which may explain the unexpected resultsdiscovered with respect to the transgenic plants disclosed herein.

In plants, most of the transport processes are energized by the primarytranslocation of protons. H⁺-translocating pumps located at the plasmamembrane and tonoplast translocated H⁺ from the cytosol to extracellularand vacuolar compartments, respectively (Rea, P. A., et al., TonoplastAdenosine Triphosphate and inorganic Pyrophosphatase. In: Methods PlantBiochem., pp. 385-405, Academic Press Limited, London (1990)). The planttonoplast contains two H⁺-translocating pumps; the V-ATPase and theinorganic pyrophosphatase or V-PPase. Their action results in luminalacidification and the establishment of a H⁺ electrochemical potentialgradient across the tonoplast (Davies, J. M., et al., The Bioenergeticsof Vacuolar H⁺ Pumps. In: Plant Vacuole, pp. 340-363, Leigh, R. A.,Sanders, D. (eds.), Academic Press, San Diego (1997)). The vacuolarmembrane is implicated in a broad spectrum of physiological processesthat include cytosolic pH stasis, compartmentation of regulatory Ca²⁺,sequestration of toxic ions such as Na⁺, turgor regulation, and nutrientstorage and retrieval. The vacuole constitute 40 to 99% of the totalintracellular volume of a mature plant cell. The vacuolar proton pumpingpyrophosphatase is a universal and abundant component of plant tonoplastcapable of generating a steady-state trans-tonoplast H⁺ electrochemicalpotential similar or greater than the one generated by the V-ATPase(Rea, P. A., et al., Tonoplast Adenosine Triphosphate and InorganicPyrophosphatase. In: Methods Plant Biochem., pp. 385-405, Academic PressLimited, London (1990)). Pyrophosphate (PPi) is a by-product in theactivation or polymerization steps of a wide range of biosyntheticpathways and in plants serves as an alternative energy donor to ATP forsucrose mobilization via sucrose synthetase, for glycolysis via PPi:fructose-6-phosphate phosphotransferase and for tonoplast energizationvia the vacuolar proton pumping pyrophosphatase (Stitt, M., Bot. Acta111:167-175 (1998)).

Most of intracellular organelles, including clathrin-coated vesicles,endosomes, Golgi membranes and vacuoles have acidic interiors (Xie, X.S., et al., J. Biol. Chem., 264:18870-18873 (1989)). This acidificationis mediated by a proton-translocating electrogenic ATPase and in plantvacuoles also via a pyrophosphate-driven proton pump V-PPase (Davies, J.M., et al., The Bioenergetics of Vacuolar H⁺ Pumps. In: Leigh R. A.,Sanders, D., (eds) The Plant Vacuole, pp. 340-363, Academic Press, SanDiego (1997); Zhen, R. G., et al., “The Molecular and Biochemical Basisof Pyrophosphate-Energized Proton Translocation at the Vacuolar MembraneAcademic Press Limited (1997)). There exists a requirement of aniontransport to maintain net electroneutrality (al-Awqati, A., Curr. Opin.Cell. Biol., 7:504-508 (1995)).

FIG. 4A is a schematic representation of a working model of thetransporters involved in sodium sequestration at the yeast pre-vacuolarcompartment; Nhx1 (Na⁺/H⁺ antiporter), Vmal (vacuolar membraneH⁺-ATPase), Gef1 (yeast CLC chloride channel), Ena1 (plasma membraneNa⁺-ATPase). The yeast member of the CLC voltage-gated chloride channelsuperfamily, Gef1, is required for copper loading in late-Golgi vesiclesand for cation sequestration in the pre-vacuolar compartment in yeast(Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA, 95:4046-4050(1998); Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA, 96:1480-1485(1999); Example 1). Furthermore, it has been shown that the defects ofgef1 mutants can be suppressed by the introduction of the prototypemember of the CLC superfamily, the Torpedo marmorata CLC-0 or by theintroduction of Arabidobsis thaliana CLC-c and CLC-d chloride channelgenes (Hechenberger, M., et al., J. Biol. Chem., 271:33632-33638 (1996);Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA, 95:4046-4050(1998)). FIG. 4B is a schematic representation of a working model of thetransporters involved in sodium sequestration at the yeast pre-vacuolarcompartment shown in FIG. 4A, which also includes Avp1 (A. thaliainavacuolar pyrophosphate-energized proton pump).

While not wishing to be bound by theory, two observations led to theproposal of the model for Na⁺ sequestration in yeast illustrated inFIGS. 4A and 4B. First, gef1 mutants are sensitive to high NaClconcentrations. Second, the Na⁺/H⁺ exchanger Nhx1 is localized to thepre-vacuolar compartment (Nass, R., et al., J. Biol. Chem.,273:21054-21060 (1998)). The model proposed in FIGS. 4A and 4B positsthat Na⁺ sequestration by Nhx1 depends on the vacuolar H⁺-ATPase andGef1, the chloride channel. Gef1-mediated anion influx allows theestablishment by the vacuolar H⁺-ATPase of a proton gradient sufficientin magnitude to drive the uphill accumulation of Na⁺ via Na⁺/H⁺exchange.

Based on such models, it was theorized that increasing the influx ofprotons into the postulated endosomal compartment should improve Na⁺sequestration via the Nhx1 exchanger. In order to increase the H⁺availability the A. thaliana gain-of-function mutant gene AVP1-D thatcodes for the vacuolar pyrophosphate-energized proton pump was expressed(FIG. 3B) (Zhen, R. G., et al., J. Biol. Chem., 272:22340-22348 (1997)).This plant pump expressed in yeast restored the Na⁺ resistance of thetest strain, but only if the strain had functional NHX1 and GEF1 genes.Furthermore, Gef1p and Nhx1p co-localize within a common organelle, thepre-vacuolar compartment (Gaxiola, R. A., et al., Proc. Natl. Acad. Sci.USA, 96:1480-1485 (1999)). These results strongly support the model inFIGS. 4A and 4B and indicate that the yeast pre-vacuolar compartment canbe used to identify the elusive plant transporters involvedintracellular sodium detoxification.

The model set forth in FIGS. 4A and 4B is entirely consistent with thephysiological data on the role of the vacuole in cation detoxificationin higher plants. Yeast and plant cells share pathways and signals forthe trafficking of vesicles from the Golgi network to the vacuole(Neuhaus, J. M., et al., Plant Mol. Biol., 38:127-144 (1998); (Paris,N., et al., Plant Physiol., 115:29-39 (1997); Sato, M. H., et al., J.Biol. Chem., 272:24530-24535 (1997); Vitale, A. V., et al., Trends PlantSci., 4:148-154 (1999)). Studies were therefore undertaken in yeast toidentify the role of the vacuole in cation detoxification in higherplants.

Studies of Cation Sequestration Mechanism in Yeast EXAMPLE 1Functionality of AtNhx1 and Avp1 in Yeast Strains

To test the sequestration models set forth in FIGS. 4A and 4B, mutantyeast strains (ena1) lacking the plasma membrane sodium efflux pump,which therefore must rely on the internal detoxification system in orderto grow on high salt, were constructed. The sequestration model (Nass,R. and Rao, R., J. Biol. Chem., 273:21054-21060 (1998) and, Gaxiola, R.A., et al., Proc. Natl. Acad. Sci. USA, 95:4046-4050 (1998)) of FIGS. 4Aand 4B predicts that the ena1 strain would become salt tolerant if onecould enhance the availability of protons in the postulated endosomalcompartment, that is with increased influx of protons, cytoplasmic Na⁺would be sequestered via the Nhx1 exchanger.

The yeast vacuolar ATPase is a multisubunit protein, so it is difficultto increase its activity by overexpressing any one of its subunits.Instead the same effect was achieved by increasing the influx of protonsby expressing the A. thaliana AVP1 gene in yeast. This gene encodes asingle polypeptide that, when expressed in yeast, is capable of pumpingprotons into the lumen of the vacuole (Kim, E. J., et al., Proc. Natl.Acad. Sci. USA, 91:6128-6132 (1994)). To ensure maximum activity of thisproton pump, the E229D gain-of-function mutant of the AVP1 gene (AVP1-D)that has enhanced H⁺ pumping capability was expressed (Zhen, R. G., etal., J. Biol. Chem., 272:22340-22348 (1997)). Intracellular sodium andpotassium content was determined for mutant and wild-type cells aftergrowth on SD-ura medium with high NaCl content.

Materials and Methods

Yeast Strains and Plasmids

Strains isogenic to W303 (ura3-1 can1-100 leu2-3, 112trp1-1 his3-11,(Gaxiola, R. A., et al., EMBO J., 11:3157-3164 (1992)) were employed.Plasmids pRG52 (*gef1::HIS3) (Gaxiola, R. A., et al., Proc. Natl. AcadSci. USA, 95:4046-4050 (1998)) and pRG197 (*nhx1::HIS3) were used toconstruct the deletions of GEF1 and NHX1 genes, yielding strains RGY85and RGY296, respectively. The ena1::HIS3 mutant was obtained from FinkLab collection (L5709).

Method of Transformation

Transformation of yeast cells was performed by using the lithium acetatemethod (Gietz, D., et al., Nucleic Acids Res., 20:1425 (1992)). Doublemutants RGY324 (gef1::HIS3 ena1::HIS3), RGY326 (nhx1::HIS3 ena1::HIS3),and RGY343 (gef1::HIS3 nhx1::HIS3) were obtained by crossing thesingle-mutant strains. Double mutants were identified among the meioticprogeny by scoring for the phenotypes associated with each of the singlemutants. Sporulation, tetrad dissection, and mating types were scored asdescribed (Guthrie C. and Fink, G. R., Guide to Yeast Genetics andMolecular Biology (Academic, San Diego (1991)). Cells were grown in YPD(1% yeast/2% peptone/2% dextrose; DIFCO), YPGAL (1% yeast/2% peptone/2%galactose; DIFCO), SD (DIFCO; Synthetic medium with 2% Dextrose), or APG(APG is a synthetic minimal medium containing 10 mM arginine, 8 mMphosphoric acid, 2% glucose, 2 mM MgSO₄, 1 mM KCl, 0.2 mM CaCl₂, andtrace minerals and vitamins) (Rodriguez-Navarro, A. and Ramos,(Rodriguez, Navarro, A. and Ramos, J., J., J. Bacteriol., 159:940-945(1984)). MnCl₂ (Sigma), tetramethylammonium chloride (Sigma), NaCl(Sigma), or hygromycin-B (Sigma were added as indicated.

Wild type, L5709 (ena1::HIS3), RGY324 (gef1::HIS3 ena1::HIS3), andRGY326 (nhx1::HIS3 ena1::HIS3) strains were transformed with pYES2vector (Invitrogen) and plasmid pYES2-AVP1-E229D described in Zhen, R.G., et al., J. Biol. Chem., 272:22340-22348 (1997). The strain RGY343(gef1::HIS3 nhx1::HIS3), used for histochemical analysis, wastransformed with pRG151 (GEF1-GFP) (Gaxiola, R. A., et al. Proc. Natl.Acad. Sci. USA, 95:4046-4050 (1998)) and with pRIN73 [NHX1-(HA)₃] (Nass,R., and Rao, R., J. Biol. Chem., 273:21054-21060 (1998)).

Wild-type and RGY296 (nhx1::HIS3) strains were transformed with vectorpAD4 (Ballester, R., et al., Cell, 59:681-686 (1989)). RGY296(nhx1::HIS3) was transformed with pRG308 (ADH1::AtNHX1) (see Cloning ofAtNHX1).

Determination of Intracellular Sodium and Potassium Content

Cells were grown overnight in SD-ura medium (DIFCO; synthetic mediumwith 2% dextrose without uracil). YPGAL (1% yeast extract/2% peptone/2%galactose; DIFCO) media was inoculated with the overnight stocks andgrow to an A600 of 0.6. At this OD, NaCl was added to a finalconcentration of 0.7 M. The cells were incubated for 6 h, harvested bycentrifugation, washed two times with 1.1 M sorbitol and 20 mM MgCl₂,and entracted with water for 30 min at 95° C.

The amount of Na⁺ and K⁺ in cells was determined at the University ofGeorgia Chemical Analysis Laboratory by an Inductively CoupledPlasma-MS. Intracellular cation concentrations were estimated asdescribed (Gaxiola, R. A., et al., EMBO J., 11:3157-3164 (1992)) byusing the intracellular water value calculated for cells grown in 1MNaCl.

Immunofluorescence

The strain RGY343 (gef1::HIS3 nhx1::HIS3) was grown in SD-ura, -leumedium (DIFCO); synthetic medium with 2% dextrose without uracil andleucin) to mid-logarithmic phase, 0.1 mg/ml hygromycin B was added, andthe culture was incubated for 1 h at 30° C. Cells were fixed with 3.7%formaldehyde (Sigma) for 45 min at room temperature without agitation.Spheroplast formation, permeablization, washing, and antibody incubationwas performed as described (Pringle, J., et al., in ImmunofluorescenceMethods for Yeast, eds. Guthrie, C. And Fink, G. F. (Academic, SandDiego), Vol. 194 pp. 565-602 (1991)). MAB HA11 used as first antibodywas from Babco (Richmond, Calif.). Cy3-conjugated goat anti-mouse IgGwas from Jackson Immunoresearch. 4′,6-Diamidino-2-phenylindole (Sigma)was added to mounting medium to stain mitochondrial and nuclear DNA.

Subcellular Fractionation and Western Analysis

The strain RGY343 (gef1::HIS3 nhx1::HIS3) was grown in APG medium (pH7.0), and lysates fractionated on a 10-step sucrose density gradient asdescribed (Nass, R. and Rao, R., J. Biol. Chem., 273:21054-21060(1998)). Aliquots of individual fractions (100 μg) were subjected toSDS/PAGE and transferred to nitrocellulose as described (Nass, R. andRao, R., J. Biol. Chem., 273:21054-21060 (1998)). Western blots wereprobed with monoclonal anti-GFP (green fluorescent protein) antibody(1:10,000 dilution; CLONTECH), anti-hemagglutinin antibody (1:10,000dilution: Boehringer Mannheim), and peroxidase-coupled goat anti-mouseantibody (1:5,000;) and developed by using the ECL enhancedchemiluminescence system (Amersham Pharmacia).

Cloning of AtNHX1

AtNHX1 was cloned from a phage cDNA library of A. thaliana (Kieber, J.J., et al., Cell, 72:427-441 (1993)) (obtained from the ArabidopsisBiological Resource Center) by probing with an expressed sequence tag(Arabidopsis Biological Resources Center, DNA Stock Center) containing apartial clone. A full-length clone (2.1 kB) was ligated into vector pSK2(Stratagene) at the Notl sit, generating plasmid pRG293. The AtNHX1 ORFwas amplified via PCR by using pRG293 as template andGGCCCGGGATGGATTCTCTAGTGTCGAAACTGCCTTCG (SEQ ID NO: 1) (italicized basescorrespond to nucleotides 1-30 of the ORF) and T7 oligonucleotides. ThePCR product was then digested with XbaI and SalI and ligated into pAD4vector generating plasmid pRG308. The AtNHX1 ORF was sequenced to verifythe fidelity of the PCR product. The full-length sequence is longer thanthe ORF reported by the: Arabidopsis Genome Initiative (A TM021B04.4),and has been deposited in GenBank (accession no. AF106324).

Cloning of AVP1-D

Vector pYES2 (Invitrogen) was introduced into wild-type, ena1, ena1nhx1, and ena1 gef1 mutants. Plasmid pYes2-AVP1-D (Zhen, R. G., et al.,J. Biol. Chem., 272:22340-22348 (1997)) was introduced into ena1, ena1nhx1, and ena1 gef1 mutants. Five-fold serial dilutions (starting at 10⁵cells) of each strain were plated on YPGAL (1% yeast extract/2%peptone/2% galactose) with or without 0.5 M NaCl and incubated at 30° C.for 2 days. Exponentially growing cells (wild-type and ena1 transformedwith pYES2 vector and ena1, ena1 nhx1, and ena1 gef1 mutants carryingpYes2-AVP1-D) were exposed to 0.7M NaCl for 6 hours. Total cell extractswere prepared, and Na⁺ and K⁺ concentrations were determined.

Results

The ena1 mutant of the above construct lacks the plasma membrane sodiumefflux pump and therefore must rely on the internal detoxificationsystem to overcome sodium toxicity. Growth of the ena1 strain issensitive to low concentrations of sodium (200 mM), concentrations thatdo not inhibit the growth of wild-type strains. Overexpression of AVP1-Drestored salt tolerance to salt-sensitive ena1 mutants. The restorationof salt tolerance to an ena1 strain by AVP1-D requires functional NHX1and GEF1 genes: ena1nhx1 AVP1-D and ena1 gef1 AVP1-D strains are saltsensitive.

FIG. 5A and FIG. 5B are bar graphs showing the intracellular Na⁺ and K⁺contents of wild-type yeast strains and of yeast strains carryingvarious mutations affecting sodium tolerance wherein the values are themean of two determinations, and the bars represent the standarddeviations. The intracellular Na⁺ and K⁺ contents of wild-type strainsand of strains carrying various mutations affecting sodium tolerancewere determined after 6 h of exposure to media supplemented with 0.7 MNaCl. The intracellular Na⁺ content in the ena1 mutant was seen to be8-fold higher than in the wild-type strain. There was seen to be aconsistent reduction in total cell Na⁺ in the ena1 AVP-D strain. Thereason for this reduction is unknown. The ena1 AVP-D strain was found tobe salt-resistant, even though its intracellular Na⁺ content was 4-foldhigher than that of the wild type. In ena1 AVP1-D strains lacking eithergef1 or nhx1 (i.e., ena1 gef1 or ena1 nhx1 ), the Na⁺ content was notreduced to the extent that it was in the GEF1 NHX1 strain. Takentogether, the genetic and physiological data are consistent with themodel that Nhx1, Gef1 and Avp1 cooperate to sequester sodium internally.As can be seen in the graphs, the Arabidopsis VacuolarH⁺-Pyrophosphatase (Avp1) was evidenced to confer salt tolerance toyeast ena1 mutants.

The intracellular K⁺ content was found to correlate with salt toleranceand is inversely correlated with the Na⁺ content of the strains (FIG.4B). The wild-type K⁺ concentration was at 100 mM, but was reduced to 20mM in the ena1 mutant. Interestingly, in an ena1 strain thatoverexpresses the AVP1-D gene, the intracellular concentration of K⁺ wasrestored almost to wild-type levels (FIG. 4B). However, AVP1-Doverexpression failed to restore wild-type levels of intracellularpotassium unless both NHx1 and GEF1 were functional (See, the doublemutants ena1 nhx1 or ena1 gef1 in FIG. 4B).

As shown herein, intracellular Na⁺ detoxification in yeast requiresfunctional Na⁺/H⁺ exchanger (Nhx1) and chloride channel (Gef1), and theyco-localize to a pre-vacuolar compartment (Gaxiola, R. A., et al., Proc.Natl. Acad. Sci. USA, 96:1480-1485 (1999)). When the Arabidopsisthaliana homologue of the yeast NHX1 gene (AtNHX1) was cloned and itsfunction in the nhx 1 yeast mutant tested, the AtNHX1 gene was found tobe able to suppress partially the cation sensitivity phenotypes of nhx1mutants.

EXAMPLE 2 Functionality of, and Co-Localization of Gef1p and Nhx1p inYeast Strains

The NHX1 and GEF1 genes, which have been identified as important insodium detoxification, are also required for the detoxification of othercations. An investigation was made with respect to the viability ofyeast strains mutant with respect to gef1 and nhx1 (ena1) in thepresence of toxic cations, in light of the model set forth in FIGS. 4Aand 4B.

The sequestration model postulates not only a functional connectionbetween the anion channel Gef1 and sodium exchanger Nhx1 but alsopredicts that these two proteins co-localize within a commoncompartment. Because previous studies indicated that Nhx1 localizes to apre-vacuolar compartment (Nass, R. and Rao, R., J. Biol. Chem.,273:21054-21060 (1998)), experiments were also performed to determinewhether Gef1 and Nhx1 proteins co-localize to this compartment.

Materials and Methods

The strain RGY419 (gef1 nhx1) was transformed with plasmids pRG151;GEF1-GFP and pRIN73; NHX1-(HA)₃. Transformants were grown in SD (DIFCO;synthetic medium with 2% dextrose). To determine the sensitivity of suchtransformants to toxic cations, five-fold serial dilutions (starting at10⁵ cells) of the indicated strains were grown at 30° C. for 2 days onYPD (1% yeast extract/2% peptone/2% dextrose) with the addition ofeither 3 mM MnCl₂, 0.45 M tetramethylammonium (TMA), or 0.05 mg/mlhygromycin B (HYG) as indicated.

Two studies were undertaken to demonstrate co-localization of Gef1p andNhx1p.

Distribution of fluorescence and immunodetection of subcellularfractions in gef1 nhx1 cells transformed with two constructs: a GEF1-GFPfusion and a NHX1-(HA)₃-tagged fusion were determined. When the cellsreached OD₆₀₀=0.5, hygromycin B (Sigma) was added to a finalconcentration of 0.1 mg/ml and the cells were incubated for 40 min at30° C. Cells were fixed and stained with antibodies to HA epitope and4′,6-diamidino-2-phenylindole (DAPI). Cells were viewed bycharge-coupled device microscopy and optically sectioned by using adeconvolution algorithm (Scanalytics, Billerica, Mass.) (Kennedy, B. K.,et al, Cell, 89:381-391 (1997)); (Bar=1·m.).

The migration properties of the Gef1p and Nhx1p in sucrose gradients wasalso determined to provide evidence of co-localization of Nhx1 (HA)₃ andGEF1-GFP. The strain RGY419 (gef1 nhx1) was transformed with plasmidspRG151; GEF1-GFP and pRIN73; NHX1-(HA)₃ and grown in APG medium(Rodriguez-Navarro, A. and Rea, P. A., J. Biol. Chem., 159:940-945(1984)). Such was converted to spheroplasts, lysed, and fractionated ona 10-step sucrose gradient (18-54%) as described (Sorin, A., et al., J.Biol. Chem., 272:9895-9901 (1997) and Antebi, A. and Fink, G. R., Mol.Biol. Cell, 3:633-654 (1992)). Western blots showed the distribution ofGef1-GFP and Nhx1-HA.

Results

Gef1 mutants were found to be sensitive to 3 mM MnCl₂, 0.45 Mtetramethylammonium chloride and to 0.05 μg/ml hygromycin-B. The nhx1mutant was also found to be sensitive to tetramethylammonium chlorideand hygromycin. The extreme sensitivity of the nhx-1 mutant tohygromycin may provide an important tool for assaying nhx1 function.

It was found that hemagglutinin (HA)-tagged Nhx1 and Gef1-GFP fusionprotein co-localize as shown via epifluorescence deconvolutionmicroscope. Persistence of signal coincidence on 90° rotation of theimage further supports co-localization of the two transporter proteinsin these cells. The co-localization of Nhx1 (HA)₃ and GEF1-GFP is alsosupported by the co-migration of the two proteins in sucrose densitygradients of membrane preparations obtained from cells expressing thetagged proteins. The sedimentation behavior of the membrane fractioncontaining both proteins is consistent with that of a pre-vacuolarcompartment (Nass, R. and Rao, R., J. Biol. Chem., 273:21054-21060(1998)). Gef1-GFP (but not Nhx1) is also present in Golgi fractions,consistent with previous studies (Gaxiola, R. A., et al., Proc. Natl.Acad. Sci. USA, 95:4046-4050 (1998), Schwappach, B., et al., J. Biol.Chem., 273:15110-15118 (1998)).

EXAMPLE 3 Capacity of A. thaliana Homolog of NHX1 to Suppress HygromycinSensitivity of Mutant Yeast

The yeast strain described herein provides an important tool foridentifying genes that mediate salt tolerance in other organisms. Totest the utility of this system, a sequence from Arabidopsis (SeeMaterials and Methods) with very high homology to the S. cerevisiae NHX1ORF was identified and used an expressed sequence tag (see Materials andMethods) to obtain a full-length clone of this Arabidopsis gene. Analignment of the amino acid sequences of Nhx1 homologues fromArabidopsis (AtNhx1), human (HsNhe6), and yeast (ScNhx1) revealssegments of amino acid identity and similarity within predictedtransmembrane domains (FIGS. 6A-C). However, it is important to notethat despite these relationships, neither the C-terminal regions ofAtNhx1 and ScNhx1 show a high degree of homology (FIGS. 6A-C).

A characteristic of mammalian Na⁺/H⁺ antiporters is their inhibition byamiloride. A putative amiloride binding site (¹⁶³DVFFLFLLPPI¹⁷³) (SEQ IDNO: 4) has been defined via point mutants in the human NHE1 antiportergene (Counillon, L., et al., Proc. Natl. Acad. Sci. USA, 90:4508-4512(1993)). AtNhx1, HsNhe-6 and ScNhx1 have an almost identical sequence(FIG. 6). However, attempts to inhibit the activity of either Nhx1 orAtNhx1 in yeast cultures with amiloride were unsuccessful.

The extreme sensitivity of yeast nhx1 mutants to hygromycin permittedthe testing of whether the cloned Arabidopsis AtNHX1 ORF could provideNa⁺/H⁺ exchange function in yeast. Vector pAD4 (Ballester, R., et al.,Cell, 59:681-686 (1989) was introduced into wild-type and nhx1 strains.Plasmid pRG308; ADH; AtNHX1 was introduced into nhx1 mutants asindicated. Five-fold serial dilutions (starting at 10⁵ cells) of theindicated strains were grown at 30° C. for 2 days on YPD (−) or on YPDsupplemented with 0.05 mg/ml hygromycin (+). Serial dilutions of thesame strains were grown on APG medium (see Materials and Methods) (−) oron APG supplemented with 0.4 M NaCl (Rodriguez-Navarro, A. and Ramos,J., J. Bacteriol., 159:940-945(1984).

The At NHX1 gene is capable of suppressing the hygromycin sensitivity ofthe nhx1 mutant. The AtNHX1 gene also suppressed the NaCl sensitivity ofnhx1 mutant but only under conditions in which the K⁺ availability wasreduced. However, AtNHX1 was not capable of rescuing the Na⁺⁻ sensitivegrowth phenotype of the double mutant ena1 nhx1 overexpressing theAVP1-D gene.

EXAMPLE 4 Generation of Gain-of-Function Yeast AtNHX Mutants

Materials and Methods

Gain of function mutants of the AtNHX that enhance salt tolerance may begenerated in the ena1 yeast by mutagenizing the cloned gene to make amutant library. This library may be used to transform the salt sensitiveyeast mutant ena1 and clones with an enhanced salt tolerant phenotype.

Other genes that show similarity to the AtNHX1 gene, as reported by theArabidopsis Genome Initiative (AGI), may also be expressed in the mutantyeast to form gain-of-function mutants. It is believed that some ofthese AtNHX1 homologues are plasma membrane transporters, so theirfunction in yeast are frequently pH dependent, requiring precisecomposition and pH of the medium used for screening for success.Identification of plasma membrane transporters helps to engineer plantswith an enhanced salt tolerance due to a reduced sodium uptake. Inaddition, plant cDNA expression libraries in yeast may be used toidentify other families of transporters involved in NaCl detoxification.

To generate gain of function mutants of the AtNHX a method forintroducing random mutations developed by Stratgene (Epicurian ColiXL1-Red competent Cells Cat#200129) may be used. The method involves thepropagation of a cloned gene into a strain deficient in the threeprimary DNA repair pathways. The random mutation rate in this strain isabout 5000-fold higher than that of wild-type. A library of the mutatedAtNHX gene may be transformed into the ena1 yeast mutant and screenedfor salt tolerance. Yeast transformation was performed as described bySchiestl and coworkers (Gietz, D., et al., Nucl. Acid Res. 20:1425(1992), incorporated by reference in its entirety herein). Analternative to the XL1-Red random mutagenesis strategy is a PCR approachdescribed by Fink and coworkers (Madhani, H. D., et al., Cell,91:673-684 (1997)).

To test AtNHX1 homologues the same strains and conditions may be asdescribed in Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA,96:1480-1485 (1999). However, others may be employed. When dealing withplasma-membrane ATNHX1 homologues pH conditions of the assay media maybe crucial.

Results

The overexpression of the A. Thaliana gain-of-function mutant geneAVP1-D increases the intracellular detoxification capability in yeast(Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA, 96:1480-1485(1999)). It is hypothesized (although the inventors are not limited bysuch hypothesis) that the latter is due to an increased influx of H⁺into the vacuolar compartment thereby improving Na⁺ sequestration viathe Nhx1 exchanger.

Conclusions from Yeast Cation Sequestration Studies

The yeast studies described above provide evidence for the importance ofthe pre-vacuolar pH for intracellular Na⁺ sequestration in yeast.Overexpression of the plant H⁺-pyrophosphatase (Avp1) confers salttolerance to yeast only in those strains containing a functionalchloride channel (Gef1) and the Na⁺/H⁺ exchanger (Nhx1).

These data support a model in which the Nhx1 Na⁺/H⁺ exchanger acts inconcert with the vacuolar ATPase and the GEF1 anion channel to sequestercations in a pre-vacuolar compartment. Several studies suggest that thepre-vacuolar compartment may be derived both from the plasma membraneand the late Golgi. These vesicles are likely involved in the assemblyof the vacuole or delivery of cargo to this organelle. It is reasonableto expect that these pre-vacuolar vesicles detoxify cations bysequestration, thereby lowering their concentrations in the cytoplasmand in other organelles.

The yeast system described herein permits the functional assessment ofdiverse heterologous proteins in salt tolerance: chloride channels, H⁺pumps, and Na⁺/H⁺ exchangers and other cation/H⁺ exchangers orcation/bicarbonate symporters. The system is robust and flexible. Thefunction of the Arabidopsis chloride channels (Gaxiola, R. A., et al,Proc. Natl. Acad. Sci. USA, 95:4046-4050 (1998), Hechenberger, M., etal., J. Biol. Chem., 271:33632-33638 (1996)), H⁺ pump, and Na⁺/H⁺exchanger can be assayed in the corresponding yeast mutant. Despite theinability of At NHX1 to suppress all the phenotypes of the yeast nhx1mutant, the fact that it suppresses some phenotypes, coupled with theDNA homology between AtNHX1 and yeast NHX1, indicates that the plantgene carries out a similar function to that of the yeast homologue. Theobservation that the AtNHX1 gene suppresses the sensitivity of the nhx1mutant to hygromycin but provides only a weak Na⁺ detoxificationphenotype could be a consequence either of differential regulation ofthe transporters in the two organisms or of distinct cation transportselectivities.

The regulation of AtNHX1 by salt and the ability of the plant gene tosuppress the yeast nhx1 mutant suggest that the mechanism by whichcations are detoxified in yeast and plants may be similar. Indeed,previous work suggested that vacuolar sodium accumulation insalt-tolerant plants may be mediated by a tonoplast Na⁺/H⁺ antiporterthat utilizes the proton-motive force generated by the vacuolarH⁺-ATPase (V-ATPase) and/or H⁺-translocating pyrophosphatase (V-Ppase;refs. Barkla, B. J., et al., Symp. Soc. Exp. Biol., 48:141-153 (1994),Zhen, R. G., et al., The Molecular and Biochemical Basis ofPyrophosphate-Energized Proton Translocation at the Vacuolar Membrane(Academic, San Diego), Kirsh, M, et al., Plant Mol. Biol., 32:543-547(1996)).

The finding described herein that both gef1 and nhx1 mutants arehypersensitive to hygromycin indicate that the level of resistance tohygromycin depends on the function of the vacuolar and pre-vacuolarorganelles. Yeast mutants impaired in K⁺ uptake (trk1) arehypersensitive to hygromycin (Madrid, R., et al., J. Biol. Chem.,273:14838-14844 (1998)); reduced K⁺ uptake hyperpolarizes the plasmamembrane potential and drives the uptake of alkali cations such ashygromycin. Mutations that reduce the H⁺ pumping activity of the plasmamembrane H⁺-ATPase, Pmal, depolarize the plasma membrane potential andconfer resistance to hygromycin (McCusker, J. H., et al., Mol. Cell.Biol., 7:4082-4088 (1987)). Thus, mutants such as gef1 or nhx1 thataffect the pH or membrane potential of the vacuolar and pre-vacuolarcompartments may be expected to affect hygromycin compartmentation.

Transgenic Plants with Unregulated Vacuolar H⁺-Translocating PumpActivity

Further support for the role of the Arabidopsis AtNHX1 gene in salthomeostasis is provided by the observation that its expression isinduced in salt-stressed plants (Gaxiola, R. A., et al., Proc. Natl.Acad. Sci. USA, 96:1480-1485 (1999)). Arabidopsis thaliana, inparticular, has been used as a host model plant; to demonstrate thatoverexpression of these genes results in salt tolerance in the plant. Arecent report shows that the overexpression of AtNHX1 gene in transgenicArabidopsis thaliana promotes sustained growth in soil watered with 200mM NaCl plus ⅛ M.S. salts under short-day cycle conditions (Apse, M., etal., Science, 285:1256-1258 (1999)). It is worth noting that everyaddition of ⅛ M.S. salts provides 2.5 mM potassium reducing thestringency of the NaCl stress, and that a short-day cycle reducesoxidative stress.

EXAMPLE 5 Enhanced Expression of At NHX1 Gene in Salt-Stressed Plants

Materials and Methods

A. thaliana plants (ecotype Columbia) were grown aseptically onunsupplemented plant nutrient agar without sucrose (Haughn, G. W. andSomerville, C., Mol. Gen. Genet., 204:430-434 (1986)) for 15 days at 19°C. and under continuous illumination. NaCl or KCl was added to a finalconcentration of 250 mM, and the plants 10 were incubated for 6 h. TotalRNA from tissue of salt-treated and untreated plants was isolated(Niyogi, K. K. and Fink, G. R., Plant Cell, 4:721-733 (1992)), HYBOND-N(Amersham) membranes were hybridized with a ³²P-Labeled DNA probe fromplasmid pRG308. Hybridization was performed at 65° C. overnight. Washeswere performed at 65° C. with 0.2% standard saline citrate (SSC)10.1%SDS (Ausebel, F., et al., Curr. Protocols in Mol. Biol. (Wiley, N.Y.)(1988)). 18S probe was used as loading control (Unifried, I., et al.,Nucleic Acids Res., 17:7513 (1989)). MACBAS 2.4 program was used toquantify the relative amount of RNA.

Results

The NaCl stress increased AtNHX1 mRNA levels 4.2-fold, whereas KClpromoted only a 2.8-fold increase. This increase in mRNA level producedby sodium resembles that described for the yeast NHX1 gene (Nass, R. andRao, R., J. Biol. Chem., 273:21054-21060 (1998)). RNA tissue blothybridized with AtNHX1. Ten micrograms of total RNA from 15-day oldplants exposed to 250 mM NaCl or KCl for 6 h and a control grown withoutsalt was subjected to electrophoresis on a denaturing formaldehyde gel.The blot was hybridized with a probe internal to AtNHX1 ORF. An 18Sribosomal probe was used as a loading control.

EXAMPLE 6 Salt Tolerance of Transgenic Plants Overexpressing AtNHX1

Transgenic plants that overexpress the AtNHX1 show sustained growth insoil watered with 200 mM sodium chloride. (Apsem M., et al., Science,285:1256-1258 (1999).

EXAMPLE 7 Salt Tolerance of Transgenic Plants Overexpressing 35SAVP1

Materials and Method

A transgenic Arabidopsis thaliana plant was engineered to overexpressthe AVP1 wild-type gene using the double tandem enhancer of the 35Spromoter (Topfer, R., et al., Nucl. Acid Res., 15:5890 (1987)). AVP1encodes the pyrophosphate-energized vacuolar membrane proton pump fromArabidopsis (Zhen, R. G., et al., J. Biol. Chem., 272:22340-22348(1997)). Previous investigations suggest that the AVP1 gene is presentin a single copy in the genome of Arabidopsis (Kim, Y., et al., PlantPhysiol., 106:375-382 (1994)), however, a sequence homologous, but notidentical, to AVP1 on chromosome one has been tentatively designated asORF F9K20.2 on BAC F9K20 by the Arabidopsis Genome Initiative (AGI).

Transgenic plants that overexpress AVP1 were generated usingAgrobacterium-mediated plant transformation. The transgenic AVP1 wasexpressed using a double tandem enhancer of the 35S promoter of CaMV(Topfer, R., et al., Nucl. Acid Res., 15:5890 (1987)). 15 wild-typeplants and 15 35SAVP1 transgenics were grown on a 24 hours-day cycle for16 days. During this period plants were watered every 4 days with adiluted nutrient solution (⅛ M.S. salts). 200 mM NaCl was added to thewatering solution at day 17 and at day 27 plants were watered withnutrient solution containing 250 mM NaCl. Plants were photographed 10days after the last NaCl treatment. Identical conditions and treatmentas described in Example 6 were used.

Results

Five different lines of 35SAVP1 plants showed an enhanced salt toleranceas compared to wild-type plants in the T2 stage. However, the mostdramatic phenotype was apparent in the homozygous T3 plants. Thesetransgenic plants are larger than wild-type plants. Furthermore,homozygous 35SAVP1 plants showed sustained growth in the presence of 250mM NaCl plus ⅛ M.S. salts when grown in a 24 hours light regimen. When35SAVP1 plants were grown under short-day cycle conditions (12 hourday/light cycle) sustained growth in the presence of 300 mM NaCl plus ⅛M.S. salts was observed.

EXAMPLE 8 Growth of Wild Type Plants and 35SAVP1 Transgenic Plants inHydroponic Solution

The reduced availability of fresh water for standard agriculture mayforce the use of alternative agricultural arts. It is conceivable thatwith salt tolerant crops the use of hydroponics with seawater willcreate a new era in crop production. Hydroponic culture has beenreported to increase plant growth and provide stress-free root and shootmaterial (Gibeaut, D. M., et al., Plant Physiol, 317-319 (1997)).Another important advantage of hydroponic culture is that it allows oneto alter the ionic composition in a more accurate manner than in soil.These advantages could be important for the physiological studies ofsalt stress.

Conditions for hydroponics culture of Arabidopsis plants wereestablished and their performance in increasing concentrations of NaClin their media were tested.

Materials and Methods

In one test, wild type and 35SAVP1 transgenic plants were hydroponicallygrown. Wild type and 35SAVP1 transgenic plants were grown in solutionculture on a 12 hour light cycle for 65 days.

In another test, wild type and 35SAVP1 transgenic plants were grown insolution culture on a 12 hours light cycle for 20 days. Starting at day21, NaCl concentration was increased in a stepwise fashion by 50 mMincrements every 4 days. Plants were photographed after 4 days in thepresence of 200 mM NaCl.

And yet in another hydroponic test, transgenic plants were challengedwith a commercial seawater formula that contains the complete ioniccomposition present in the oceans. 35SAVP1, 35SAtNHX1 single and doubletransgenics were grown together with wildtype Arabidopsis thalianaplants under hydroponic conditions for four weeks in a short dayillumination cycle (Gibeaut, D. M., et al., Plant Physiol., 317-319(1997)). Then every four days an equivalent to 50 mM NaCl of TROPICMARIN sea salt is added. This artificial sea water mix includes all ofthe other major and trace elements present in real sea water. Growth wasmonitored and physiological parameters, such as sodium content anddistribution may be monitored.

Results

When wild type and 35SAVP1 transgenic plants were grown hydroponicallythe size differences in root, leaves and stems among wild type and35SAVP1 transgenic plants were found to be dramatic, with 35SAVP1transgenic plant parts being much larger.

When NaCl concentration were increased stepwise by 50 mM every 4 days(Apse, M., et al., Science, 285:1256-1258 (1999), 35SAVP1 transgenicplants appeared healthy in the presence of 200 mM NaCl while wild typecontrols showed severe deleterious effects in their leaves and stems.

35SAVP1, 35SAtNHX1 single and double transgenics that were growntogether with wildtype Arabidopsis thaliana plants under hydroponicconditions for four weeks in a short day illumination cycle (Gibeaut, D.M., et al., Plant Physiol., 317-319 (1997)) and then challenged everyfour days with an equivalent to 50 mM NaCl of TROPIC MARIN sea salt werefound to grow in the sea salt solution better than wildtype Arabidopsisthaliana plants.

EXAMPLE 9 Effect of Overexpression of Arabidopsis thaliana ProtonTransporters in Tomato Plants

The effects of the overexpression of these Arabidopsis thaliana protontransporters (AVP1 and AtNHX1) in a more agriculturally important plant,the tomato plant, may be examined. It is believed that increasing thesalt-tolerance of tomato plants will likely have important economicrepercussions.

The tomato homologues of AVP1 and AtNHX1 may be isolated and thecorresponding chimeras to overexpress them may be constructed (Bidone,S., et al., Eur. J. Biochem., 253: 20-26 (1998); Burbidge, A., et al.,J. Exper. Botany, 48:2111-2112 (1997)). The genes may be introduced viaAgrobacterium-mediated infection of calli. Tissue culture methods may beused to regenerate transformed plants. The plants may be assayed forsalt tolerance as well as physiological parameters, such as sodiumcontent and distribution.

Tomato transformation with 35S AVP1 and with 35S AtNHX1 constructs maybe performed as described by McCormick (McCormick, S., Transformation oftomato with Agrobacterium tumefaciens. In: Plant Tissue Culture Manual,pp. 1-9, Lindsey, K. (ed.), Kluwer Academic Publishers, Dordrecht, TheNetherlands (1991)). T0 and T1 transgenics may be analyzed by polymerasechain reaction and DNA gel blotting for the presence and copy number ofAVP1 and ATNHX1 transgenes. Heterozygous and homozygous plants may beidentified after segregation analysis of each transgenic within T1seeds. Homozygous plants may be assayed for salt tolerance and as wellas physiological parameters, such as sodium content and distribution.Degenerated oligos based on conserved sequences present in AVP1 andAtNHX1 homologues may be designed. These degenerated primers may be usedin RT-PCR reactions with cDNAs made from poly(A)+RNA from tomato. Theresulting PCR fragments may be used as probes to isolate the full lengthcDNA clones from commercial libraries (i.e. STRATAGENE Cat#936004). Asimilar strategy was described by Caboche and coworkers (Quesada, A., etal., Plant Mol. Biol., 34:265-274 (1997)).

Results

Positive test results would indicate that the sequestration modeldescribed herein is also applicable to an important crop.

FIG. 7 is a graph of Na⁺ and K⁺ content of wild-type plants (WT) versusrepresentative transgenic plants overexpressing AVP-1 (1′ and 2′) grownin salty soil. Five wild-type plants (WT) and two AVP-1 overexpressingtransgenic lines (1′ and 2′) were grown on soil in a 10 hour light/darkcycle. Plants were watered with a diluted nutrient solution (⅛ MS salts)for six weeks and subsequently watered with a diluted nutrient solutionsupplemented with NaCl. The concentration of NaCl began with 100 mM andwas increased every four days by 50 mM. The photograph corresponds toplants at the tenth day in the presence of 300 mM NaCl. Parts of theplant above, ground were harvested after 24 hours in the presence of 200mM NaCl and their fresh weigh measured. After 48 hours at 75° C., thedry weight was measured. Na⁺ and K⁺ content was determined by atomicabsorption. Values in the graphs of FIG. 4 are the mean +/− SE (n=4). Ascan be seen from the graphs Na⁺ and K⁺ content in the transgenic lines(1′ and 2′) was significantly higher than that of wild-typecounterparts.

FIG. 8 is a graph of the uptake of calcium into the 35SAVP-1 transgenicvacuolar membrane vesicles (squares) of 2′ of FIG. 4 versus calciumuptake into vesicles obtained from wild type (WT) of FIG. 4. Wild-typeplants (open circles) and transgenic plants from line 2′ of FIG. 4 weregrown hydroponically for nine weeks on a 10 hour light cycle. Vacuolarmembrane vesicles were added to buffer containing 250 mM sorbitol, 25 mMBTP-Hepes pH 8.0, 50 mM KCl, 1.5 nM MgSO₄ and 10 μM Ca⁺⁺. This mix wasincubated at 20° C. for 10 minutes before adding 200 μM PPi to triggerthe reaction. Ca⁺⁺ ionophore A23187 was added to a final concentrationof 5 μg/ml to dissipate the Ca⁺⁺ gradient. Aliquot (200 μl) werefiltered at the indicated times and washed with cold buffer as described(11). As is evidenced by the graphs, the transgenic plants from line 2′have greater calcium uptake than wild-type plants.

The above data is consistent with the hypothesis that transgenic plantsoverexpressing AVP-1 have an enhanced H⁺ pumping capability at theirtonoplast and that an enhanced H⁺ supply results in greater ionaccumulation in the vacuole through the action of H⁺-driven iontransporters. To further support this theory, Ca⁺⁺ uptake capability ofwild type and transgenic vacuolar membrane vesicles was determined.

It is well documented that Ca⁺⁺ enters the plant vacuole via a Ca⁺⁺/H⁺antiporter (K. S. Schumaker, H. Sze, Plant Physiol. 79, 1111-1117(1985)). Furthermore, the genes encoding the Arabidopsis thalianaCa⁺⁺/H⁺ antiporters CAX1 and CAX2 have been isolated and characterized(K. D. Hirschi, R. -G. Zhen, K. W. Cunningham, P. A. Rea, G. R. Fink,Proc. Natl. Acad. Sci. USA 93, 8782-8786 (1996)). FIG. 8 shows that Ca⁺⁺uptake in the 35SAVP-1 transgenic vacuolar membrane vesicles is 36%higher than it is in vesicles obtained from wild type. Application ofthe Ca⁺⁺ ionophore A23 lowered the 45Ca⁺⁺ counts to background levelsdemonstrating the tightness of the vesicles (FIG. 8) (K. S. Schumaker,H. Sze, Plant Physiol 79, 1111-1117 (1985)).

While not limited by such theory, a model consistent with the enhanceddrought and freeze tolerance of the transgenic plants overexpressing theAVP-1 gene is depicted in FIGS. 9A and 9B. The model depicts how anincrease in the number of AVP-1 pumps in the vacuole of transgenicplants can provide more H⁺ that will permit the secondary transportersto import greater amounts of cations into the lumen of the vacuoles.Higher amounts of cations confer a greater osmotic pressure (See, FIG.6) that leads to a greater water retention capability endowing plants towithstand low soil water potentials.

EXAMPLE 10 Double Transgenic Plant with 35SAVP1 and 35S AtNHX1

Overexpression of the pyrophosphate-energized vacuolar membrane protonpump AVP1 likely increases the availability of H⁺ in the lumen of thevacuole, and the AtNHX1 Na⁺/H⁺ antiporter uses these H⁺ to sequester Na⁺cations into the vacuole. Therefore, higher expression of thesetransporters likely maximizes the sequestration capability of thevacuole.

To generate transgenic Arabidopsis plants that overexpress both genesAVP1 and AtNHX1, T3 35S AVP1 plants may be used as females and T3 35SAtNHX1 plants may be used as males. Female plants may behand-emasculated and anthers from freshly opened flowers of donor plantsare harvested. With these anthers the emasculated plants may bepollinated by touching the anthers onto the stigmas. The pollinatedflowers may be labeled and any remaining opened or unopened flowers fromthe same female plant removed to avoid any confusion at harvest. Theharvested seeds should be sterilized using a 50% sodium hypochloridesolution and mixed vigorously for 5 minutes and rinsed with waterthoroughly. The sterilized seeds may be stored in soft agar over nightat 4° C. Then they may be sprinkled onto solidified kanamycin-hygromycinselective medium. The 35S AVP1 construct has the neomycinphosphotransferase II gene that confers kanamycin tolerance in plantswhile the 35S AtNHX1 construct has a modified hygromycin Bphosphotransferase that confers hygromycin tolerance in plants. Theresistant seedlings may be transplanted into soil and to the hydroponicmedia to be tested for their salt-tolerant phenotype.

A transgenic Arabidopsis thaliana plant to overexpress the A. thialianagain-of-function mutant gene AVP1-D (Zhen, et al., J. Biol. Chem.,272:22340-22348 (1997)) may be engineered using the same double tandemenhancer of the 35A promoter described above (Topfer, R., et al., Nucl.Acid Res., 15:5890 (1997)). Plants overexpressing the gain of functionmutant gene will likely show an enhanced phenotype. These plants amay becharacterized in parallel with the 35SAVP1, 35S AtNHX singles anddoubles trangenics. The A. thaliana gain-of-function mutant gene AVP1-Dmay be subcloned into plasmid pRT103 carrying the 35S promoter and thepolyadenylation signal of CaMV (Topfer, R., et al., Nucl. Acid Res.,15:5890 (1997)). A HindIII fragment containing the chimeric 35SAVP-Dgene may be subcloned into pBIBhyg (Becker, D., Nucl. Acid Res., 18:203(1990)). The resulting T-DNA vector may be transformed intoAgrobacterium tumefaciens strain GV3101 via electroporation, and may beused for subsequent vacuum infiltration of Arabidopsis thaliana ecotypeColumbia (Bechtold, N., et al., C. R. Jeances Acad. Sci. Ser. III Sci.Vie, 316:1194-1199 (1993)). Integration may be confirmed on Southernblots of T3 plants and expression monitored on Northern blots ofpositive T3 plants.

EXAMPLE 11 Comparative Transport Study with Vacuoles from the Roots ofWild-Type and 35S AVP1 Transgenic Plants

A study may be undertaken to determine if the vacuoles of 35S AVP1transgenic plants show a higher proton transport activity dependent onpyrophosphate. These determinations may be done with root and shoottissues separately from plants grown hydroponically. The transgene couldshow a tissue-specific regulation despite the 35S promoter.

In order to compare the PPI-dependent H⁺ translocation activities ofwild-type and 35S AVP1 transgenic plants sealed tonoplast-enrichedvesicles from roots and leaves of the above plants may be prepared. Thehomogenization and differential centrifugation procedure described byRea and Turner (Rea, P. A., et al., Tonoplast Adenosine Triphosphate andInorganic Pyrophosphatase. In: Meth. Plant Biochem., pp. 385-405,Academic Press limited, London (1990)) may be followed. H⁺ translocationmay be assayed fluorimetrically using acridine orange (2.5 μM) astransmembrane pH difference indicator in assay media containing vacuolemembrane-enriched vesicles as described by Rea and coworkers (Zhen, R.G., et al., J. Biol. Chem., 272:22340-22348 (1997)). The assay mediacontains 300-μM Tris-PPi, 50 mK KCl, 2.5 μM acridine orange, 5 mMTris-Mes (pH 8.0). Intravesicular acidification may be triggered withthe addition of 1.3 mM MgSO4 and terminated with the addition of theprotonophore FCCP at 2.5 μM. Fluorescence may be measured at excitationemission wavelengths of 495 and 540 nM, respectively, at a slit width of5 nM (Zhen, R. G., et al., J. Biol. Chem., 269:23342-23350 (1994)). Afurther test to support that the H⁺ translocation is AVP1 driven may bethe addition of the specific inhibitor aminomethylenediphosphonate(Zhen, R. G., et al., Plant Physiol., 104:153-159 (1994)).

EXAMPLE 12 Determination of the Na⁺/K⁺ Ratios in Leaves and Stems of theTransgenic Plants

Toxic concentrations of NaCl build up first in the fully expanded leaveswhere NaCl is compartmentalized in the vacuoles. Exposure to NaCl candisrupt or reduce K⁺ uptake leading to K⁺ deficiency and growthinhibition (Wu, S. J., et al., Plant Cell, 8:617-627 (1996). A cytosolicconsequence of reduced K⁺ content and high Na⁺ is the inhibition ofimportant enzymes. An example of such enzymes is the 3′(2′),5′-bisphosphate nucleotidase of yeast whose activity is more sensitiveto Na⁺ when K⁺ content is low (Murguia, J. R., et al., Science,267:232-234 (1995).

Measurements may be taken to demonstrate that the transgenic plantsdescribed herein have an increased vacuolar capacity to sequester Na⁺ intheir leaves cells or elsewhere. To determine the Na⁺/K⁺ ratios inleaves and stem S wild-type and 35S AVP1/35S AtNHX1 double and singletransgenics in hydroponic conditions (Gibeaut, D. M., et al., PlantPhysiol., 317-319 (1997) may be grown. NaCl may be added to the growthmedia in a stepwise fashion starting with 50 mM up to 250 mM (Apse, M.,et al., Science, 285:1256-1258 (1999). At every point the rosette andthe stems of the treated plants may be collected and their weightdetermined. The samples should be dried out in an oven at 80° C. andtheir dry weight determined. The dry samples may be boiled in adetermined volume of water and their Na⁺ and K⁺ contents determined viaatomic absorption spectrophotometry (Apse, M., et al., Science,285:1256-1258 (1999); Gaxiola, R., etal., Embo J., 11:3157-2164 (1992)).

EXAMPLE 13 Determination of Whether 35S AVP1 Transgenic Plants AreLarger Because Their Cells Are Larger or Because They Have More Cells,or Both

The shoot meristems labeling index may be compared with one of thewild-type plants. Morphological and anatomical observations measuringand counting cells of leaves, roots and stems may be performed. Todetermine if 35S AVP1 transgenic plants are larger because they havemore cells, their shoot meristems labeling index may be compared withthe one of wild-type plants.

To measure the DNA synthesis or cell proliferation5-Bromo-2′-deoxy-uridine (BrdU) that can be incorporated into DNA inplace of thymidine may be used. Cells that have incorporated BrdU intoDNA may be detected using a monoclonal antibody against BrdU monoclonalantibody and an anti-mouse Ig-alkaline phosphatase as a second antibody.The bound anti-BrdU monoclonal antibody may be visualized by lightmicroscopy and the ratio between DAPI stained and BrdU positivesestablished. The protocol is a modification of the one published byChiatante and coworkers (Levi, M., et al., Physiol. Plant. 71:68-72(1987)) and the BrdU labeling and detection kit II from BoehringerMannheim. The plants may be exposed for different times to the BrdUlabeling medium and then fixation, paraffin embedding and sectioning maybe performed as described by Meyerowitz and coworkers (Drews, G., etal., Plant Mol. Biol. Rep., 5:242-250 (1988)).

For observation of leaf tissue, fresh tissues may be embedded in 5%agarose and slice them with a microslicer. For primary root observation,seedlings may be fixed for 4 hr in 50% ethanol, 5% acetic acid, and 3.7%formaldehyde at room temperature, dehydrated in graded ethanol series,permeate them with xylene, and infiltrate them with paraffin.Eight-micrometer sections may be stained with 0.05% toluidine blue andcells may be counted under a microscope. As an alternative for thevisualization and determination of cell size the method described byGreenberg and coworkers (Rate, et al., The Plant Cell, 11:1695-1708(1999)) may be followed.

Results

Microscopic studies indicate that the cells of the transgenicArabidopsis plants are not larger, but that the number of cells isgreater in the transgenics versus wild-type. Macroscopically it was seenthat in the AVP-1 line-2′ an average of eight more leaves in the rosettewas noted over wild-type. F1 plants originated by crossing transgeniclines 1′ and 2′ displayed rosettes with larger leaves and increasedamount of leaves than wild-type plants. FIG. 10 depicts the foliage ofwild type and transgenic (1′ and 2′) Arabidopsis plants overexpressingAVP-1 grown at 20° C. under all white fluorescent light in 16 hourslight/8 hours dark period cycle. Leaves depicted were carefully sectoredwith a scalpel when plants initiated to bolt and then ordered by sizefor comparison purposes. While having larger and more leaves than thewild-type plants, the transgenics, were not seen to have larger cellsizes. Such data is consistent with the hypothesis that the meristem ismore active in transgenic AVP-1 overexpressers.

Dry weight of the transgenic Arabidiopsis plants grown hydroponically,as compared to similarly grown wild-type plants, further indicates thatcell mass increases irrespective any increased water uptake by theplant. An increase in dry mass weight was seen in both the root, rosetteand stem structures as indicated in Table 1 below where values representthe mean values of six plants.

TABLE 1 Dry Weight of Arabidopsis Plant Parts Grown HydroponicallyWILD-TYPE PLANT PART (WT) 1′ TRANSGENIC 2′ TRANSGENIC Roots 0.03 g 0.05g 0.14 g Rosette 0.10 g 0.20 g 0.60 g Stems 0.40 g 0.50 g 0.80 g

EXAMPLE 14 Effect of Overexpression of AVP-1 on Hormonal Activity

Increased meristematic activity and/or shoot organogenesis may beproduced by increasing hormone availability together with AVP-1overexpression.

The effect of overexpression of AVP-1 on hormone activity was adjudgedplacing true leaves from wild type (WT) and transgenic (1′, 2′)Arabidopsis plants overexpressing AVP-1 by careful sectoring with ascalpel. Leaves were placed on 5 layers of filter paper saturated withdistilled water in Petri dishes. The Petri dishes, were incubated at 20°C. under cool white fluorescent light in a 16 h light/8 h darkcycles. Asseen in FIGS. 11A (view of leaves on the petri dishes) and 11B (view ofleaves from petri dishes of FIG. 11A positioned so as to more clearlyset depict root structure developing form the leaves), leaves fromtransgenic plants remain greener and had an enhanced rooting capability.An enhanced rooting capability suggests increased auxin content anddelayed senescence consistent with increased cytokinin levels.

EXAMPLE 15 Effect of Overexpression of AVP-1 on Callus Induction inPetunia Explants Method

Petunia explants were incubated on MS medium which consists of MS salts(Gibco BRL), 1 mg/L nicotinic acid, 1 mg/L pyrodoxin HCl, 1 mg/Lthiamine, 100 mg/L myo-inositol, 3% sucrose, 1 mg/L2,4-D(2,4-dichlorophenoxyacetic acid) and 0.5 mg/L 6-BA(6-benzylaminopurine). The medium was solidified with 0.7% agar and wasadjusted to pH 5.8 before autoclave. The culture was incubated at 25° C.in the dark in a growth chamber.

Results

Transgenic petunia explants (35-S AVP-1) demonstrated significantlyenhanced callus induction at 6 weeks of incubation as demonstrated inFIG. 15.

EXAMPLE 16 Effect of Overexpression of AVP-1 on Shoot Regeneration FromLeave Segments

Leave segments grown in appropriate medium are known to be capable ofgenerating shoot growth. A study was undertaken to determine the effectof overexpression of 35-S AVP-1 on shoot regeneration in Petunia leaves.

Segments of leaves from regenerated transformed (35-S AVP-1) and controlPetunia were used as explants for shoot regeneration. The leaves werecut with a sharp surgical blade into about 1 cm wide pieces. Theexplants were cultured in MS medium which included MS salts (Gibco), B5vitamins (1 mg/L nicotinic acid, 1 mg/L pyrodoxin HCl, 1 mg/L-thiamineand 100 mg/L myo-inositol), 3% sucrose, 2 mg/L 6-benzylaminopurine and0.01 mg/L napthaleneacitic acid, 0.7% agar, pH 5.8. The culturedsegments were incubated at 25° C. under cool white fluorescent light ina 16 h light/8 h dark period cycle.

As shown in FIG. 12, petunia leaves transformed with the AVP-1 gene(35-S AVP-1 petri dish) under a constitutive promoter show an enhancedshoot regeneration capability over wild-type petunia leave segment (WT)similar to results with transgenic Arabidopsis plants. These results areconsistent with the idea that overexpression of this vacuolar protonpump will improve the shoot regeneration capacity of any plant.

A blast search using AVP-1 ORF as a probe showed this gene is highlyconserved (rice=86% identities; tobacco=89% identities; barley=86%identities; Vitis vinifera (grapes)=82% identities, Hordeun vulgare(barley)=86% identities and Zea mays (corn)=90% identities). The latterindicates that overexpression of the AVP-1 gene would similarly causesuch effects in other plant types.

EXAMPLE 17 Effect of Overexpression of AVP-1 on Shoot and RootRegeneration from Arabidopsis Cotyledons

With appropriate medium, it is known that shoots and roots may beregenerated from cotyledons. Shoot and root regeneration from cotyledonswas adjudged for wild-type (WT) and AVP-1 transgenic Arabidopsis (1′ and2′).

Five (5) day old cotyledons were used as explants for regeneration.Explants were incubated on shoot induction medium (SIM) at 20° C. under16 h light/8 h dark period. The SIM contained MS salts (Gibco), B5vitamins including 1 mg/L nicotinic acid, 1 mg/L pyrodoxin HCl, 1 mg1-thiamine, and 100 mg/L myoinositol, 3% sucrose, 1 mg/L6-(gama-gama-dimethylallylamino)purine riboside (2-iP) and 0.1 mg/Lnaphtaleneacitic acid and solidified with 0.7% agar. The pH of themedium was adjusted to 5.8. 2-iP was filter-sterilized and added to themedium after autoclave.

As is evidenced in the pictures of FIGS. 13A and 13B, explants fromtransgenic plants (1′ and 2′) regenerate shoots and roots at a higherfrequency and earlier than wild type (WT), consistent with a highermeristematic competence. The evidence of difference between thetransformed and wild-type cotyledons was dramatically evidenced at day23 (FIG. 13A) and more so 37 days into culturing (FIG. 13B).

EXAMPLE 18 Effect of Overexpression of AVP-1 on Shoot Regeneration fromRoot and Cotyledon Explants in Arabidopsis Plants

Root and cotyledon (5 days old) explants from wild-type (WT) andtransgenic (1′ and 2′) AVP-1 overexpressing Arabidopsis plants wereplaced in the shoot induction medium as described in Example. 16. Asevidenced in FIG. 6, explants from the transgenic plants (1′ and 2′)generated new structure earlier than wild type consistent with a highermerstematic compentence.

EXAMPLE 19 Isolation of Mutants in the Transporters

Genetic approaches are very powerful in analyzing complex biologicaltraits (Serrano, R., Crit. Rev. Plant Sci., 13:121-138 (1994)) Reversegenetics is a very important new tool for plant biologists. Thegeneration of a good collection of tagged knockouts by Sussman andcoworkers (Krysan, P., et al., Proc. Natl. Acad. Sci. USA, 93:8145-8150(1996)) has open a very important avenue for the analysis of genedisruptions in Arabidopsis.

The Arabidopsis Knock-out Facility of the University of WisconsinMadison (world wide web atbiotech.wisc.edu/NewServicesAndResearch/Arabidopsis) may be used tosearch among the 60,480 Arabidopsis (ecotype WS) lines that have beentransformed with the T-DNA vector pD991 for the presence of T-DNAinserts within AtCLC-c, AtCLC-d, AVP1, AtNHX1 and their homologues. Thephenotypes of the above knock-outs will shed light towards theunderstanding of the physiological roles of these transporters in normaland stress conditions. An initial characterization of the knockoutplants includes testing for their salt tolerance and their Na⁺/K⁺ratios. The generation of double knock-outs via crosses help to furtherunderstand the interaction among the transporters as well as the crosseswith the 35S AVP1 and the 35S ATNHX1 transgenic plants.

To search for Arabidopsis knock-out PCR primers may be designedfollowing the guidelines detailed in the University of Wisconsin website. Tested primers may be sent to UW-Madison, where 62 PCR reactionsthat are sent to us for Southern blot analysis may be performed.Positive PCR products are sequenced. If the sequence reveals that thereis a T-DNA inserted within the gene the gene specific primers are sentfor another set of PCR reactions in order to determine which of the 9possible pools of 225 contains the knockout. After identifying the poolof interest, 25 tubes of seeds are screened for the individual plantcarrying the T-DNA knock-out.

EXAMPLE 20 Cation Detoxification in Plant Cells

The studies described herein together with other evidence stronglyindicate that yeast and plants share pathways and signals for thetrafficking of vesicles from Golgi network to the vacuole (Gaxiola, R.,et al., Proc. Natl. Acad. Sci. USA, 96:1480-1485 (1999); Marty, F., “TheBiogenesis of Vacuoles: Insights from Microscopy. In: The Plant Vacuole,1-42, Leigh, R. A. and Sanders, D., Academic Press, San Diego (1997);Bassham, D. C., et al., Plant Physiol, 117:407-415 (1998)).

Without wishing to be bound by theory, it is believe likely by thepresent inventor that in both systems a pre-vacuolar compartment is adynamic entity that detoxifies the cytoplasm from toxic cations anddelivers its cargo either to the vacuole, or directly to the cellexterior. Both the gef1 chloride channel and Nhx1 Na⁺/H⁺ exchanger havebeen localized to the yeast pre-vacuolar compartment (Gaxiola, R. A., etal., Proc. Natl. Acad. Sci. USA, 96:1480-1485 (1999)). The behavior ofthe gef1-GFP chimera in yeast cells in vivo have been monitoredindicating that its localization varies depending the environmentalconditions. Furthermore, it has been shown that two of the four A.thaliana CLC chloride channel genes CLC-c and -d are capable ofsuppressing gef1 mutant phenotypes implying a similar localization(Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA, 95:4046-4050(1998)).

In order to understand how and where this cation detoxification takesplace in plant cells the intracellular localization of GFP chimeras ofAVP1, AtNHX1 and AtCLC-c and -d (Hong, B., et al., Plant Physiol,119:1165-1175 (1999)) may be monitored in vivo. Confocal microscopy maybe also used to address co-localization of the different transporters.For this purpose HA-tagged versions or antibodies of the transportersunder study are required (Guiltinan, M. J., et al., Meth. Cell Biol.,49:143-151 (1995); Jauh, G. -Y., et al., Plant Cell, 11:1867-1882(1999); Mullen, R. T., et al., Plant. J., 12:313-322 (1997)).

For the constructions of the GFP-chimeras the soluble versions GFP withimproved fluorescence in A. thaliana reported by Davis and Viestra(Davies, S. J., Viestra, R. D., “Soluble derivatives of greenfluorescent protein (GFP) for use in Arabidopsis, thaliana,http://brindabella.mrc-lmb.cam.ac.uk/IndexGFP.html (1998)). may be used.Two types of GFP-chimeras may be made, namely a set under the regulationof the native promoter and another set under the regulation of the 35Spromoter. The resulting T-DNA vectors containing the GFP-chimeras aretransformed into Agrobacterium tuniefaciens strain GV3101 viaelectroporation, and used for subsequent vacuum infiltration ofArabidopsis thaliana ecotype Columbia (Bechtold, N., et al., C. R.Jeances Acad. Sci. Ser. III Sci. Vie, 316:1194-1199 (1993)). For thehemagglutinin (HA) epitope tagging a PCR strategy designed for yeast butmodified to tag plant genes expressed in yeast vectors may be used.Futcher and coworkers designed vectors containing the URA3 yeast geneflanked by direct repeats of epitope tags (HA) (Schneider, B. L., et al,Yeast, 11:1265-1274 (1995)). Via PCR the tag-URA3-tag cassette may beamplified such that the resulting PCR fragment possess homology at eachend to the gene of interest., In vivo recombination in yeast can be thenused to direct the integration of the PCR-chimera to the plasmidcarrying the plant ORF of interest, transformants are selected by theURA⁺ phenotype. The URA3 gene can be “popped out” when positivetransformants are grown in the presence of 5-fluoro-orotic acid. Thevector carrying the plant gene has a selection marker different than theURA3 gene.

In conclusion, the manipulation of vacuolar V-PPases in economicallyimportant crops could provide an important avenue for crop improvement.Drought and freeze tolerant cultivars could provide new agriculturalapproaches in areas lost due to drought or minimal rainfall, as well asto provide farmers with protection from unanticipated frosts (freezingrain etc.). Such crops may also be able to be raised on soils consideredtoo saline for wild type crops.

While the invention has been described with respect to preferredembodiments, those skilled in the art will readily appreciate thatvarious changes and/or modifications can be made to the inventionwithout departing from the spirit or scope of the invention as definedby the appended claims. All references cited in this specification areherein incorporated by reference to the same extent as if eachindividual reference was specifically and individually indicated to beincorporated by reference.

1. A method of making a transgenic plant with one or more enhancedphenotypic traits relative to non-transgenic wild-type plants of thesame species, wherein the one or more enhanced phenotypic traits areselected from the group consisting of: increased biomass in one or moreplant parts, increased shoot regeneration, increased root regeneration,and enhanced callus induction, said method comprising: a) introducingexogenous nucleic acid encoding a plant vacuolar pyrophosphatase intocells of a plant to generate transformed cells, wherein the exogenousnucleic acid is operably linked to at least one regulatory element thatcauses overexpression of plant vacuolar pyrophosphatase in thetransformed cells; b) regenerating transgenic plants from thetransformed cells; and c) selecting for a transgenic plant with one ormore enhanced phenotypic traits relative to non-transgenic wild-typeplants of the same species, wherein the one or more enhanced phenotypictraits are selected from the group consisting of: increased biomass oneor more plant parts, increased shoot regeneration, increased rootregeneration, and enhanced callus induction.
 2. The method of claim 1,wherein the transgenic plant is selected from the group consisting oftomato, rice, tobacco, sorghum, cucumber, lettuce, turf grass,Arabidopsis, corn, petunia, barley and grape.
 3. The method of claim 2,wherein the cells of a plant are obtained from a tissue selected fromthe group consisting of roots, stems, leaves, flowers, fruits and seeds.4. The method of claim 3, wherein the exogenous nucleic acid encoding aplant vacuolar pyrophosphatase is obtained from a plant selected fromthe group consisting of Arabidopsis, tobacco, rice, barley, grape andcorn.
 5. The method of claim 4, wherein the regulatory element isselected from the group consisting of a tissue-specific promoter, aconstitutive promoter, an inducible promoter, and a promoter that isboth tissue-specific and inducible.
 6. The method of claim 4, whereinthe exogenous nucleic acid encoding a plant vacuolar pyrophosphatase isoperably linked to a double tandem enhancer of a 35S CaMV promoter. 7.The method of claim 6, wherein the exogenous nucleic acid encoding aplant vacuolar pyrophosphatase is at least 82% identical to a nucleicacid encoding the Arabidopsis AVP1 protein of SEQ ID NO:2.
 8. The methodof claim 7, wherein the plant vacuolar pyrophosphatase is Arabidopsisvacuolar pyrophosphatase AVP1.
 9. The method of claim 1, wherein thetransgenic plant has increased biomass in one or more plant parts. 10.The method of claim 9, wherein the transgenic plant has increasedbiomass in one or more plant parts and the increased biomass in one ormore plant parts is selected from the group consisting of thicker stem,increased root structure, or a combination thereof.
 11. The method ofclaim 10, wherein the transgenic plant has increased root structure andthe increased root structure comprises longer root hairs.
 12. The methodof claim 9, wherein the transgenic plant has increased biomass of thewhole plant.
 13. A method of making a transgenic plant with increasedbiomass in one or more plant parts, said method comprising: a)introducing exogenous nucleic acid encoding Arabidopsis vacuolarpyrophosphatase AVP1 or a homolog thereof with vacuolar pyrophosphataseactivity into cells of a plant to generate transformed cells, whereinthe exogenous nucleic acid is operably linked to at least one regulatoryelement that causes overexpression of AVP1 or a homolog thereof withvacuolar pyrophosphatase activity in the transformed cells; b)regenerating transgenic plants from the transformed cells; and c)selecting for a transgenic plant with increased biomass in one or moreplant parts relative to non-transgenic wild-type plants of the samespecies.
 14. The method of claim 13, wherein the transgenic plant isselected from the group consisting of tomato, rice, tobacco, sorghum,cucumber, lettuce, turf grass, Arabidopsis, corn, petunia, barley andgrape.
 15. The method of claim 14, wherein the cells of a plant areobtained from a tissue selected from the group consisting of roots,stems, leaves, flowers, fruits and seeds.
 16. The method of claim 15,wherein the regulatory element is selected from the group consisting ofa tissue-specific promoter, a constitutive promoter, an induciblepromoter, and a promoter that is both tissue-specific and inducible. 17.The method of claim 15, wherein the exogenous nucleic acid encodingArabidopsis vacuolar pyrophosphatase AVP1 is operably linked to a doubletandem enhancer of a 35S CaMV promoter.
 18. The method of claim 13,wherein the transgenic plant has increased biomass of the whole plant.19. The method of claim 18, wherein said increased biomass of the wholeplant is measured by comparing dry weight biomass of said transgenicplants to dry weight biomass of non-transgenic wild-type plants of thesame species.